Compositions, kits and methods for identification, assessment, prevention and therapy of cancer

ABSTRACT

The invention relates to compositions, kits, and methods for detecting, characterizing, preventing, and treating human cancer. A variety of chromosomal regions (MCRs) and markers corresponding thereto, are provided, wherein alterations in the copy number of one or more of the MCRs and/or alterations in the amount, structure, and/or activity of one or more of the markers is correlated with the presence of cancer.

RELATED APPLICATIONS

This application claim the benefit of U.S. provisional application No.60/654,227, filed Feb. 17, 2005 and U.S. provisional application No.60/690,064, filed Jun. 13, 2005; the contents of each application isincorporated herein in its entirety by this reference.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, by NationalInstitutes of Health (NIH) under grants K08AG 2400401, RO1CA099041,R01CA84628, P01CA95616 and U01CA84313. The government may therefore havecertain rights to this invention.

SEQUENCE LISTING

The instant application contains a “lengthy” Sequence Listing which hasbeen submitted via CD-R in lieu of a printed paper copy, and is herebyincorporated by reference in its entirety. Said CD-R, recorded on Feb.17, 2006, are labeled “CRF”, “Copy 1”, “Copy 2”, and “Copy 3”respectively, and each contains only one identical 2.03 MB file(DFS06425.txt).

BACKGROUND OF THE INVENTION

Cancer represents the phenotypic end-point of multiple genetic lesionsthat endow cells with a full range of biological properties required fortumorigenesis. Indeed, a hallmark genomic feature of many cancers,including, for example, lung cancer, breast cancer, ovarian cancer, andcolon cancer, is the presence of numerous complex chromosome structuralaberrations—including non-reciprocal translocations, amplifications anddeletions.

Karyotype analyses (Johansson, B., et al. (1992) Cancer 69, 1674-81;Bardi, G., et al. (1993) Br J Cancer 67, 1106-12; Griffin, C. A., et al.(1994) Genes Chromosomes Cancer 9, 93-100; Griffin, C. A., et al. (1995)Cancer Res 55, 2394-9; Gorunova, L., et al. (1995) Genes ChromosomesCancer 14, 259-66; Gorunova, L., et al. (1998) Genes Chromosomes Cancer23, 81-99), chromosomal CGH and array CGH (Wolf M et al. (2004)Neoplasia 6(3)240; Kimura Y, et al. (2004) Mod. Pathol. 21 May (epub);Pinkel, et al. (1998) Nature Genetics 20:211; Solinas-Toldo, S., et al.(1996) Cancer Res 56, 3803-7; Mahlamaki, E. H., et al. (1997) GenesChromosomes Cancer 20, 383-91; Mahlamaki, E. H., et al. (2002) GenesChromosomes Cancer 35, 353-8; Fukushige, S., et al. (1997) GenesChromosomes Cancer 19:161-9; Curtis, L. J., et al. (1998) Genomics 53,42-55; Ghadimi, B. M., et al. (1999) Am J Pathol 154, 525-36; Armengol,G., et al. (2000) Cancer Genet Cytogenet 116, 133-41), fluorescence insitu hybridization (FISH) analysis (Nilsson M et al. (2004) Int J Cancer109(3):363-9; Kawasaki K et al. (2003) Int J Mol. Med. 12(5):727-31) andloss of heterozygosity (LOH) mapping (Wang Z C et al. (2004) Cancer Res64(1):64-71; Seymour, A. B., et al. (1994) Cancer Res 54, 2761-4; Hahn,S. A., et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al. (1996)Genes Chromosomes Cancer 17, 88-93) have identified recurrent regions ofcopy number change or allelic loss in various cancers. For example, inlung cancer, frequent gains have been mapped to 1q31, 3q25-27, 5p13-14and 8q23-24 and losses to 3p21, 8p22, 9p21-22, 13q22, and 17p12-13(Bjorkqvist, A. M., et al. (1998) Br J Cancer 77, 260-9; Luk, C., et al.(2001) Cancer Genet Cytogenet 125, 87-99; Pei, J., et al. (2001) GenesChromosomes Cancer 31, 282-7; Petersen, I., et al. (1997) Cancer Res 57,2331-5; Balsara, B. R. & Testa, J. R. (2002) Oncogene 21, 6877-83). Insome instances, validated oncogenes and tumor suppressor genes residingwithin these loci have been identified, including MYC (8q24),p16^(INK4A) (9p21), RB1 (13q14), RASSF1 (3p21), TUSC2 (3p21), SEMA3B(3p21), FHIT (3p14), and KRAS2 (12p12). However, for the majority ofamplified and deleted loci and resident genes, the presumedcancer-relevant targets remain to be discovered.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the identificationof specific regions of the genome (referred to herein as minimal commonregions (MCRs)), of recurrent copy number change which are containedwithin certain chromosomal regions (loci) and are associated withcancer. These MCRs were identified using a novel cDNA or oligomer-basedplatform and bioinformatics tools which allowed for the high-resolutioncharacterization of copy-number alterations in the lung cancer genome(see Example 1). The present invention is based, also in part, on theidentification of markers residing within the MCRs of the invention,which are also associated with cancer.

Accordingly, in one aspect, the present invention provides methods ofassessing whether a subject is afflicted with cancer or at risk fordeveloping cancer, comprising comparing the copy number of an MCR in asubject sample to the normal copy number of the MCR, wherein the MCR isselected from the group consisting of the MCRs listed in Tables 3 and/or7, and wherein an altered copy number of the MCR in the sample indicatesthat the subject is afflicted with cancer or at risk for developingcancer. In one embodiment, the copy number is assessed by fluorescent insitu hybridization (FISH). In another embodiment, the copy number isassessed by quantitative PCR (qPCR). In yet another embodiment, the copynumber is assessed by FISH plus spectral karotype (SKY). In stillanother embodiment, the normal copy number is obtained from a controlsample. In yet another embodiment, the sample is selected from the groupconsisting of tissue, whole blood, serum, plasma, buccal scrape, saliva,cerebrospinal fluid, urine, stool, bronchoalveolar lavage, and lungtissue.

In another aspect, the invention provides methods of assessing whether asubject is afflicted with cancer or at risk for developing cancercomprising comparing the amount, structure, and/or activity of a markerin a subject sample, wherein the marker is a marker which resides in anMCR listed in Tables 3 and/or 7, and the normal amount, structure,and/or activity of the marker, wherein a significant difference betweenthe amount, structure, and/or activity of the marker in the sample andthe normal amount, structure, and/or activity is an indication that thesubject is afflicted with cancer or at risk for developing cancer. Inone embodiment, the marker is selected from the group consisting of themarkers listed in Tables 1 or 2. In another embodiment, the amount ofthe marker is determined by determining the level of expression of themarker. In yet another embodiment, the level of expression of the markerin the sample is assessed by detecting the presence in the sample of aprotein corresponding to the marker. The presence of the protein may bedetected using a reagent which specifically binds with the protein. Inone embodiment, the reagent is selected from the group consisting of anantibody, an antibody derivative, and an antibody fragment. In anotherembodiment, the level of expression of the marker in the sample isassessed by detecting the presence in the sample of a transcribedpolynucleotide or portion thereof, wherein the transcribedpolynucleotide comprises the marker. In one embodiment, the transcribedpolynucleotide is an mRNA or cDNA. The level of expression of the markerin the sample may also be assessed by detecting the presence in thesample of a transcribed polynucleotide which anneals with the marker oranneals with a portion of a polynucleotide wherein the polynucleotidecomprises the marker, under stringent hybridization conditions.

In another embodiment, the amount of the marker is determined bydetermining copy number of the marker. The copy number of the MCRs ormarkers may be assessed by comparative genomic hybridization (CGH),e.g., array CGH. In still another embodiment, the normal amount,structure, and/or activity is obtained from a control sample. In yetanother embodiment, the sample is selected from the group consisting oftissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinalfluid, urine, stool, bronchoalveolar lavage, and lung tissue.

In another aspect, the invention provides methods for monitoring theprogression of cancer in a subject comprising a) detecting in a subjectsample at a first point in time, the amount and/or activity of a marker,wherein the marker is a marker which resides in an MCR listed in Tables3 and/or 7; b) repeating step a) at a subsequent point in time; and c)comparing the amount and/or activity detected in steps a) and b), andtherefrom monitoring the progression of cancer in the subject. In oneembodiment, the marker is selected from the group consisting of themarkers listed in Tables 1 or 2. In another embodiment, the sample isselected from the group consisting of tissue, whole blood, serum,plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool,bronchoalveolar lavage, and lung tissue. In still another embodiment,the sample comprises cells obtained from the subject. In yet anotherembodiment, between the first point in time and the subsequent point intime, the subject has undergone treatment for cancer, has completedtreatment for cancer, and/or is in remission.

In still another aspect, the invention provides methods of assessing theefficacy of a test compound for inhibiting cancer in a subjectcomprising comparing the amount and/or activity of a marker in a firstsample obtained from the subject and maintained in the presence of thetest compound, wherein the marker is a marker which resides in an MCRlisted in Tables 3 and/or 7, and the amount and/or activity of themarker in a second sample obtained from the subject and maintained inthe absence of the test compound, wherein a significantly higher amountand/or activity of a marker in the first sample which is deleted incancer, relative to the second sample, is an indication that the testcompound is efficacious for inhibiting cancer, and wherein asignificantly lower amount and/or activity of the marker in the firstsample which is amplified in cancer, relative to the second sample, isan indication that the test compound is efficacious for inhibitingcancer in the subject. In one embodiment, the first and second samplesare portions of a single sample obtained from the subject. In anotherembodiment, the first and second samples are portions of pooled samplesobtained from the subject. In one embodiment, the marker is selectedfrom the group consisting of the markers listed in Tables 1 or 2.

In yet another aspect, the invention provides methods of assessing theefficacy of a therapy for inhibiting cancer in a subject comprisingcomparing the amount and/or activity of a marker in the first sampleobtained from the subject prior to providing at least a portion of thetherapy to the subject, wherein the marker is a marker which resides inan MCR listed in Tables 3 and/or 7, and the amount and/or activity ofthe marker in a second sample obtained from the subject followingprovision of the portion of the therapy, wherein a significantly higheramount and/or activity of a marker in the first sample which is deletedin cancer, relative to the second sample, is an indication that the testcompound is efficacious for inhibiting cancer and wherein asignificantly lower amount and/or activity of a marker in the firstsample which is amplified in cancer, relative to the second sample, isan indication that the therapy is efficacious for inhibiting cancer inthe subject. In one embodiment, the marker is selected from the groupconsisting of the markers listed in Tables 1 or 2.

Another aspect of the invention provides methods of selecting acomposition capable of modulating cancer comprising obtaining a samplecomprising cancer cells; contacting said cells with a test compound; anddetermining the ability of the test compound to modulate the amountand/or activity of a marker, wherein the marker is a marker whichresides in an MCR listed in Tables 3 and/or 7, thereby identifying amodulator of cancer. In one embodiment, the marker is selected from thegroup consisting of the markers listed in Tables 1 or 2. The cells maybe isolated from, e.g., an animal model of cancer, a cancer cell line,e.g., a lung cancer cell line originating from a lung tumor, or from asubject suffering from cancer.

Yet another aspect of the invention provides methods of selecting acomposition capable of modulating cancer comprising contacting a markerwith a test compound; and determining the ability of the test compoundto modulate the amount and/or activity of a marker, wherein the markeris a marker which resides in an MCR listed in Tables 3 and/or 7, therebyidentifying a composition capable of modulating cancer. In oneembodiment, the marker is selected from the group consisting of themarkers listed in Tables 1 or 2. In another embodiment, the methodfurther comprises administering the test compound to an animal model ofcancer. In still another embodiment, the modulator inhibits the amountand/or activity of a gene or protein corresponding to a marker set forthin Table 2 which is amplified, e.g., a marker selected from the markerslisted in Table 2. In yet another embodiment, the modulator increasesthe amount and/or activity of a gene or protein corresponding to amarker set forth in Table 1 which is deleted, e.g., a marker selectedfrom the markers listed in Table 1.

In another aspect, the invention provides kits for assessing the abilityof a compound to inhibit cancer comprising a reagent for assessing theamount, structure, and/or activity of a marker, wherein the marker is amarker which resides in an MCR listed in Tables 3 and/or 7. In oneembodiment, the marker selected from the group consisting of the markerslisted in Tables 1 or 2.

The invention also provides kits for assessing whether a subject isafflicted with cancer comprising a reagent for assessing the copy numberof an MCR selected from the group consisting of the MCRs listed inTables 3 and/or 7, as well as kits for assessing whether a subject isafflicted with cancer, the kit comprising a reagent for assessing theamount, structure, and/or activity of a marker. In one embodiment, themarker selected from the group consisting of the markers listed inTables 1 or 2.

In another aspect, the invention provides kits for assessing thepresence of human cancer cells comprising an antibody or fragmentthereof, wherein the antibody or fragment thereof specifically bindswith a protein corresponding to a marker, wherein the marker is a markerwhich resides in an MCR listed in Tables 3 and/or 7. In one embodiment,the marker selected from the group consisting of the markers listed inTables 1 or 2.

In still another aspect, the invention provides kits for assessing thepresence of cancer cells comprising a nucleic acid probe wherein theprobe specifically binds with a transcribed polynucleotide correspondingto a marker, wherein the marker is a marker which resides in an MCRlisted in Tables 3 and/or 7. In one embodiment, the marker selected fromthe group consisting of the markers listed in Tables 1 or 2.

In yet another aspect, the invention provides methods of treating asubject afflicted with cancer comprising administering to the subject amodulator of the amount and/or activity of a gene or proteincorresponding to a marker, wherein the marker is a marker which residesin an MCR listed in Tables 3 and/or 7. In one embodiment, the markerselected from the group consisting of the markers listed in Tables 1 or2.

The invention also provides methods of treating a subject afflicted withcancer comprising administering to the subject a compound which inhibitsthe amount and/or activity of a gene or protein corresponding to amarker which resides in an MCR listed in Tables 3 and/or 7 which isamplified in cancer, e.g., a marker selected from the markers listed inTable 2, thereby treating a subject afflicted with cancer. In oneembodiment, the compound is administered in a pharmaceuticallyacceptable formulation. In another embodiment, the compound is anantibody or an antigen binding fragment thereof, which specificallybinds to a protein corresponding to the marker. For example, theantibody may be conjugated to a toxin or a chemotherapeutic agent. Instill another embodiment, the compound is an RNA interfering agent,e.g., an siRNA molecule or an shRNA molecule, which inhibits expressionof a gene corresponding to the marker. In yet another embodiment, thecompound is an antisense oligonucleotide complementary to a genecorresponding to the marker. In still another embodiment, the compoundis a peptide or peptidomimetic, a small molecule which inhibits activityof the marker, e.g., a small molecule which inhibits a protein-proteininteraction between a marker and a target protein, or an aptamer whichinhibits expression or activity of the marker.

In another aspect, the invention provides methods of treating a subjectafflicted with cancer comprising administering to the subject a compoundwhich increases expression or activity of a gene or proteincorresponding to a marker which resides in an MCR listed in Tables 3and/or 7 which is deleted in cancer, e.g., a marker selected from themarkers listed in Table 1, thereby treating a subject afflicted withcancer. In one embodiment, the compound is a small molecule. Theinvention also includes methods of treating a subject afflicted withcancer comprising administering to the subject a protein correspondingto a marker, e.g., a marker selected from the markers listed in Table 1,thereby treating a subject afflicted with cancer. In one embodiment, theprotein is provided to the cells of the subject, by a vector comprisinga polynucleotide encoding the protein. In still another embodiment, thecompound is administered in a pharmaceutically acceptable formulation.

The present invention also provides isolated proteins, or fragmentsthereof, corresponding to a marker selected from the markers listed inTables 1 or 2.

In another aspect, the invention provides isolated nucleic acidmolecules, or fragments thereof, corresponding to a marker selected fromthe markers listed in Tables 1 or 2.

In still another aspect, the invention provides isolated antibodies, orfragments thereof, which specifically bind to a protein corresponding toa marker selected from the markers listed in Tables 1 or 2.

In yet another aspect, the invention provides an isolated nucleic acidmolecule, or fragment thereof, contained within an MCR selected from theMCRs listed in Tables 3 and/or 7, wherein said nucleic acid molecule hasan altered amount, structure, and/or activity in cancer. The inventionalso provides an isolated polypeptide encoded by the nucleic acidmolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the genomic profiles of primary lung adeno- and squamouscarcinomas and lung cancer cell lines. The upper panel shows therecurrence of chromosomal alterations. Integer-value recurrence of CNAsin segmented data (Y axis) is plotted for each probe evenly alignedalong the X axis in chromosome order. Dark bars denote gain or loss ofchromosome material, light bars represent probes within regions ofamplification or deletion. Asterisks identify the most frequent regionof gains and losses, as reported in the literature (Balsara, B. R., andTesta, J. R. (2002) Oncogene 21, 6877-6883). The lower panel shows aheat map plot showing discrete CNAs within all samples with the X axisrepresenting probes ordered by genomic map positions and the Y axisrepresenting individual samples. Light gray represents chromosomalgain/amplification and dark gray denotes chromosomal loss/deletion.

FIG. 2 depicts the chromosomal segment length distribution. Overalllevel of genomic complexity was estimated for adenocarcinoma andsquamous cell carcinoma aCGH profiles by quantifying the number ofnarrow, high amplitude copy number alterations in each set. Within thetwo sample classes, each profile was reviewed for highly altered regionsof the genome (segments showing absolute log₂ copy number >0.4, see theMaterials and Methods section). These were plotted in a histogramaccording to the length of the region, in megabases. The area under thecurves is normalized to the overall incidence of high-level alterationper sample in the adenocarcinoma and squamouscell carcinoma datasets.

FIG. 3 show that Chromosome 3q, from 180 MB to 199 MB, is the onlygenomic region that shows significantly difference between AC and SCC byboth aCGH and expression profiling. On both plots, x-axis coordinatesrepresent probes ordered by genomic map positions, from chromosome 1 tochromosome X. (A) Probes significantly gained/amplified comparing SCCand AC primary tumors, on array-CGH. Y-axis represents the −log₁₀ Pvalue of the permutation test. Probes presenting a P<0.05 are plotted inred, probes with a P>0.05 in gray. No probes were significantlylost/deleted after comparison between SCCs and ACs primary tumors. (B)Genomic regions significantly enriched for differentially expressedprobes comparing SCC and AC primary tumors. Y-axis represents the −log₁₀adjusted p values of Fisher's exact test for enrichment. Red lineshighlight regions with an adjusted P<0.05 adjusted for multiple testing,probes with an adjusted P>0.05 are in gray.

FIGS. 4A-4F depict the verification and boundary delimitation of the8p12-p11.2 amplicon. (A). Real time PCR verification of the 8p12-p11.2amplicon and its boundaries. Two primary tumors were used to delimit theboundaries, PT3 (dark gray bars) and PT1 (light gray bars). BAC #1: BACRP11-265K5; BAC #2: RP11-100B16. Dotted square includes the genesinvolved in the amplicon. The dotted vertical line identifies the genedosage threshold above which copy number was considered increased(2-fold). (B). (Upper panel) Metaphase FISH analysis of the cell lineNCI-H1703 showing multiple light gray signals on one derivativechromosome (Light gray arrow and left inset. Light gray signal, BAC#2).A normal copy of chromosome 8 is also evident (dark gray arrow and rightinset) showing two signals for BAC#2 (light gray) and 2 signals for thecontrol BAC (RP11-138J2, telomeric to the amplicon, at 27 MB, on chr.8,dark gray signal). (Lower panel) Interphase FISH analysis on a sectionfrom the primary tumor PT3, comparing adjacent normal and tumor tissues.Note multiple, high-intensity signals with BAC#1 (light gray) in thetumor part and single or double signals with BAC#1 in the normal part ofthe section. Control BAC (RP11-138J2, is telomeric to the amplicon, at27 MB, on chr.8, dark gray signal). (C). Interphase FISH analysis on atissue section from the primary tumor PT3, showing multiplehigh-intensity signals when using BAC#1 (light gray). Dark gray arrow,control BAC (RP11-138J2, telomeric to the amplicon, at 27 MB, on chr.8,red signal). (D). Interphase FISH analysis on a tissue section from theprimary tumor PT3, showing two signals per cell, for both BAC#2 (darkgray signal, white arrow) and control BAC (RP11-138J2, telomeric to theamplicon, at 27 MB, on chromosome 8, light gray signal, light grayarrow). (E). Interphase FISH analysis on a tissue section from theprimary tumor PT5, showing multiple high-intensity signals when usingBAC#2 (dark gray signal). White arrow, control BAC (RP11-138J2,telomeric to the amplicon, at 27 MB, on chr.8, light gray signal). (F).Interphase FISH analysis on a tissue section from the primary tumor PT5,showing two signals per cell, for both BAC#3 (dark gray signal, darkgray arrow) and control BAC (RP11-138J2, telomeric to the amplicon, at27 MB, on chr.8, light gray signal, white arrow).

FIG. 5 depicts expression analyses of genes residing in the chromosome8p12-p11.2 amplicon. Heat map representation of the expression level ofeach of the genes in the amplicon from Affymetrix analysis. Whenmultiple Affymetrix probes for a gene were present, the median valueamong the probes was used. Each column represents an individual sampleand each row represents a gene. The color intensity on the heat-mapcorrelates with the intensity of the expression. The MCR positivesamples include 4 out of 5 squamous primary tumors demonstrating the8p12-p11.2 amplicon and NCI-H1703 and NCI-H520, the two lung cancer celllines with the 8p12-p11.2 amplicon. Normal represents 3 independentnormal RNA references, isolated from adjacent histologically normal lungtissues, MCR negative tumors are 2 primary tumors not showingamplification at 8p12-p11.2.

FIG. 6 depicts expression analyses of genes residing in the chromosome20q11 amplicon. Heat map representation of the expression level of eachof the 5 genes, ID1, COX412, BCL2L1, TPX2 and MYLK2, in the ampliconfrom Affymetrix analysis. When multiple Affymetrix probes for a genewere present, the median value among the probes was used. Each columnrepresents an individual sample and each row represents a gene. Thecolor intensity on the heat-map correlates with the intensity of theexpression. The arrows indicate the samples that present amplification.

FIG. 7 depicts the high level of concordance between the cDNA andoligo-aCGH profiles. A representative example is shown contrasting cDNA-and oligo-aCGH profiles for the adenocarcinoma cell line NCI-H1623. Theprofiles are depicted in light gray (cDNA-aCGH) and dark gray(oligo-aCGH) The higher resolution of the oligonucleotide array platformrelative to cDNA arrays uncovered several additional highly focal CNAs(asterisks).

FIG. 8 depicts the recurrence of chromosome 3 alterations. Integer-valuerecurrence of CNAs in segmented data (Y axis) is plotted for each probealigned along the X axis in chromosome order. Dark gray bars denote lossof chromosome material, light gray bars represent probes within regionsof deletion (see the Materials and Methods section). White spacesrepresent gaps between probes.

FIG. 9 depicts FISH analysis of the 8p12-p11.2 amplicon in the cell lineNCI-H520. In addition to two normal copies of chromosome 8 (each showingsingle-probe labeling for BAC#2, light gray, and control BAC, dark red),three additional marker chromosomes showed signals for BAC#2 (lightgray; arrows).

FIGS. 10A-10C depict FISH analysis of the 8p12-p11.2 amplicon in atissue section from the primary tumor PT1, comparing adjacent tumor andnormal tissues. In A, H&E stained slide showing normal and adjacenttumor at 20× magnification. B: 40× magnification. C: FISH of the regionhighlighted in B, including both normal tissue (inset, dotted line) andtumor tissue. High-level amplification is evident using the BAC#2 (lightgray; arrows) in the tumor but not in the normal tissue (inset in thedotted line).

FIG. 11 depicts FISH analysis of the 8p12-p11.2 amplicon in a sectionfrom the primary tumor PT2 (left) and PT4 (right). Multiplehigh-intensity signals are evident when using the BAC#2 (dark gray;arrow) when compared with the control BAC (light gray; arrowhead).

BRIEF DESCRIPTION OF THE TABLES

Table 1 contains markers of the invention which reside in MCRs ofdeletion and display decreased expression. Table 1 contains SEQ ID NOs:1-67.

Table 2 contains markers of the invention which reside in MCRs ofamplification and display increased expression. Table 2 contains SEQ IDNOs: 68-272.

Table 3 contains high-confident focal MCRs. Table 3 contains SEQ ID NOs:273-340.

Table 4 contains a description of the cell lines provided herein.

Table 5 contains a description of the tumor samples provided herein.

Table 6 contains the oligonucleotide sequences provided herein. Table 6contains SEQ ID NOs: 341-712.

Table 7 contains a list of high-confidence MCRs in lung adenocarcinoma(AC) and squamous cell carcinoma (SCC) primary tumors and cell lines.Table 7 contains SEQ ID NOs: 713-770.

Table 8 contains markers that are overexpressed in squamous cellcarcinomas (SCCs) versus adenocarcinomas (ACs) and were overexpressed inthe absence of gene copy number gains on 3q in SCCs. Table 8 containsSEQ ID NOs: 771-778.

Table 9 contains markers of the invention displaying increasedexpression in lung cancer primary tumors. Table 9 contains SEQ ID NOs:779-848.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the identificationof specific regions of the genome (referred to herein as minimal commonregions (MCRs)), of recurrent copy number change which are containedwithin certain chromosomal regions (loci) and are associated withcancer. These MCRs were identified using a novel cDNA or oligomer-basedplatform and bioinformatics tools which allowed for the high-resolutioncharacterization of copy-number alterations in the lung cancer genome,e.g., the non-small cell lung cancer (NSCLC) genome (see Example 1).

To arrive at the identified loci and MCRs, array comparative genomichybridization (array-CGH) was utilized to define copy number aberrations(CNAs) (gains and losses of chromosomal regions) in lung cancer celllines and tumor specimens.

Segmentation analysis of the raw profiles to filter noise from thedataset (as described by Olshen and Venkatraman, Olshen, A. B., andVenkatraman, E. S. (2002) ASA Proceedings of the Joint StatisticalMeetings 2530-2535; Ginzinger, D. G. (2002) Exp Hematol 30, 503-12;Golub, T. R., et al. (1999) Science 286, 531-7; Hyman, E., et al. (2002)Cancer Res 62, 6240-5; Lucito, R., et al. (2003) Genome Res 13,2291-305) was performed and used to identify statistically significantchangepoints in the data.

Identification of loci was based on an automated computer algorithm thatutilized several basic criteria as follows: 1) segments above 0.4 orbelow −0.4 (0.5 and 0.55 for cDNA) are identified as altered; 2) if twoor more altered segments are adjacent in a single profile or separatedby less than 500 KB, the entire region spanned by the segments isconsidered to be an altered span; 3) highly altered segments or spansthat are shorter than 20 MB are retained as “informative spans” fordefining discrete locus boundaries. Longer regions are not discarded,but are not included in defining locus boundaries; 4) informative spansare compared across samples to identify overlapping groups ofpositive-value or negative-value segments; each group is called an“overlap group”; 5) overlap groups are divided into separate groupswherever the recurrence rate falls below 25% of the peak recurrence forthe whole group; recurrence is calculated by counting the number ofsamples with alteration at high threshold (0.4, −0.4); and 6) minimalcommon regions (MCRs) are defined as contiguous spans having at least75% of the peak recurrence. If two MCRs are separated by a gap of onlyone probe position, they are joined. If there are more than 3 MCRs in alocus, the whole region is reported as a single complex MCR.

A locus-identification algorithm was used that defines informative CNAson the basis of size and achievement of a high significance thresholdfor the amplitude of change. Overlapping CNAs from multiple profileswere then merged in an automated fashion to define a discrete “locus” ofregional copy number change, the bounds of which represent the combinedphysical extend to these overlapping CNAs. Each locus was characterizedby a peak profile, the width and amplitude of which reflect the contourof the most prominent amplification or deletion for that locus.Furthermore, within each locus, one or more minimal common regions(MCRs) were identified across multiple tumor samples, with each MCRpotentially harboring a distinct cancer-relevant gene targeted for copynumber alteration across the sample set.

The locus-identification algorithm defined discrete MCRs within thedataset which were annotated in terms of recurrence, amplitude of changeand representation in both cell lines and primary tumors. These discreteMCRs were prioritized based on four criteria that emphasize recurrenthigh-threshold changes in both primary tumors and cell lines (seeExample 1). Implementation of this prioritization scheme yielded 126MCRs of the present invention that satisfied at least three of the fourcriteria (see Tables 3 and 7; Table 7 shows high-confidence MCRs; Table3 shows high-confidence focal MCRs) in lung adenocarcinoma and squamouscell carcinoma primary tumors and cell lines. The numbers of primarytumors (T) or cell lines (C) with gain or loss, and amplification ordeletion, are listed, respectively. MCR recurrence is denoted as apercentage of the total dataset. In bold are the MCRs verified by RT-PCRand FISH. The short arm of chromosome 3 was consistently lost acrossseveral primary tumors and cell lines (FIG. 1 and FIG. 8). Three smallerregions within the short arm of chromosome 3 were identified by theautomated locus definition program, based on the presence and recurrenceof deletions in a subset of samples (FIG. 8, bright green bars).

The confidence-level ascribed to these prioritized loci was furthervalidated by real-time quantitative PCR (QPCR), which demonstrated 100%concordance with 17 selected MCRs defined by array-CGH.

The MCRs identified herein possess a median size of 1.42 Mb, with 29(23%) MCRs spanning 0.5 Mb or less, and possess an average of 4annotated genes.

Also in Tables 3 and 7, the loci and MCRs are indicated as having either“gain and amplification” or “loss and deletion,” indicating that eachlocus and MCR has either (1) increased copy number and/or expression or(2) decreased copy number and/or expression, or deletion.

Complementary expression profile analysis of a significant fraction ofthe genes residing within the MCRs of the present invention provided asubset of markers with statistically significant association betweengene dosage and mRNA expression. Table 1 lists the markers of theinvention which reside in MCRs of deletion and which consequentlydisplay decreased expression by comparison across lung cancer celllines. Table 2 lists the markers of the invention which reside in MCRsof amplification that are overexpressed by comparison, across lungcancer cell lines. Additional markers within the MCRs that have not yetbeen annotated may also be used as markers for cancer as describedherein, and are included in the invention.

The novel methods for identifying chromosomal regions of altered copynumber, as described herein, may be applied to various data sets forvarious diseases, including, but not limited to, cancer. Other methodsmay be used to determine copy number aberrations as are known in theart, including, but not limited to oligonucleotide-based microarrays(Brennan, et al. (2004) In Press; Lucito, et al (2003) Genome Res.13:2291-2305; Bignell et al. (2004) Genome Res. 14:287-295; Zhao, et al(2004) Cancer Research, 64(9):3060-71), and other methods as describedherein including, for example, hybridization methods (such as, forexample, FISH and FISH plus spectral karyotype (SKY)).

The amplification or deletion of the MCRs identified herein correlatewith the presence of cancer, e.g., lung cancer and other epithelialcancers. Furthermore, analysis of copy number and/or expression levelsof the genes residing within each MCR has led to the identification ofindividual markers and combinations of markers described herein, theincreased and decreased expression and/or increased and decreased copynumber of which correlate with the presence of cancer, e.g., lungcancer, e.g., NSCLC in a subject.

Accordingly, methods are provided herein for detecting the presence ofcancer in a sample, the absence of cancer in a sample, and othercharacteristics of cancer that are relevant to prevention, diagnosis,characterization, and therapy of cancer in a subject by evaluatingalterations in the amount, structure, and/or activity of a marker. Forexample, evaluation of the presence, absence or copy number of the MCRsidentified herein, or by evaluating the copy number, expression level,protein level, protein activity, presence of mutations (e.g.,substitution, deletion, or addition mutations) which affect activity ofthe marker, or methylation status of any one or more of the markerswithin the MCRs (e.g., the markers set forth in Tables 1 and 2), iswithin the scope of the invention.

Methods are also provided herein for the identification of compoundswhich are capable of inhibiting cancer, in a subject, and for thetreatment, prevention, and/or inhibition of cancer using a modulator,e.g., an agonist or antagonist, of a gene or protein marker of theinvention.

Although the MCRs and markers described herein were identified in lungcancer samples, the methods of the invention are in no way limited touse for the prevention, diagnosis, characterization, therapy andprevention of lung cancer, e.g., the methods of the invention may beapplied to any cancer, as described herein.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “tumor” or “cancer” refer to the presence of cells possessingcharacteristics typical of cancer-causing cells, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Cancer cells are often in the form of a tumor, but such cells may existalone within an animal, or may be a non-tumorigenic cancer cell, such asa leukemia cell. As used herein, the term “cancer” includes premalignantas well as malignant cancers. Cancers include, but are not limited to,lung cancer, e.g., non-small cell lung carcinoma (NSCLC), melanomas,breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreatic cancer, stomach cancer, ovarian cancer, urinarybladder cancer, brain or central nervous system cancer, peripheralnervous system cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like.

The term “lung cancer” or “neoplasia” as used herein, includes non-smallcell lung carcinoma (NSCLC) and small cell carcinoma. Lung cancer may be“metastatic” from another source (e.g., colon) or may be “primary” (atumour of lung cell origin).

As used herein, the term “non-small cell lung carcinoma”, “non-smallcell lung cancer”, or “NSCLC” is a lung cancer that comprises all lungcarcinomas except small cell carcinoma, and is intended to includeadenocarcinoma (AC) of the lung, large cell carcinoma, alveolar cellcarcinoma, and squamous cell carcinoma (SCC).

A “minimal common region (MCR),” as used herein, refers to a contiguouschromosomal region which displays either gain and amplification(increased copy number) or loss and deletion (decreased copy number) inthe genome of a cancer. An MCR includes at least one nucleic acidsequence which has increased or decreased copy number and which isassociated with a cancer. The MCRs of the instant invention include, butare not limited to, those set forth in Tables 3 and 7.

A “marker” is a gene or protein which may be altered, wherein saidalteration is associated with cancer. The alteration may be in amount,structure, and/or activity in a cancer tissue or cancer cell, ascompared to its amount, structure, and/or activity, in a normal orhealthy tissue or cell (e.g., a control), and is associated with adisease state, such as cancer. For example, a marker of the inventionwhich is associated with cancer may have altered copy number, expressionlevel, protein level, protein activity, or methylation status, in acancer tissue or cancer cell as compared to a normal, healthy tissue orcell. Furthermore, a “marker” includes a molecule whose structure isaltered, e.g., mutated (contains an allelic variant), e.g., differs fromthe wild type sequence at the nucleotide or amino acid level, e.g., bysubstitution, deletion, or addition, when present in a tissue or cellassociated with a disease state, such as cancer.

The term “altered amount” of a marker or “altered level” of a markerrefers to increased or decreased copy number of a marker or chromosomalregion, e.g., MCR, and/or increased or decreased expression level of aparticular marker gene or genes in a cancer sample, as compared to theexpression level or copy number of the marker in a control sample. Theterm “altered amount” of a marker also includes an increased ordecreased protein level of a marker in a sample, e.g., a cancer sample,as compared to the protein level of the marker in a normal, controlsample. Furthermore, an altered amount of a marker may be determined bydetecting the methylation status of a marker, as described herein, whichmay affect the expression or activity of a marker.

The amount of a marker, e.g., expression or copy number of a marker orMCR, or protein level of a marker, in a subject is “significantly”higher or lower than the normal amount of a marker or MCR, if the amountof the marker is greater or less, respectively, than the normal level byan amount greater than the standard error of the assay employed toassess amount, and preferably at least twice, and more preferably three,four, five, ten or more times that amount. Alternately, the amount ofthe marker or MCR in the subject can be considered “significantly”higher or lower than the normal amount if the amount is at least abouttwo, and preferably at least about three, four, or five times, higher orlower, respectively, than the normal amount of the marker or MCR.

The “copy number of a gene” or the “copy number of a marker” refers tothe number of DNA sequences in a cell encoding a particular geneproduct. Generally, for a given gene, a mammal has two copies of eachgene. The copy number can be increased, however, by gene amplificationor duplication, or reduced by deletion.

The “normal” copy number of a marker or MCR or “normal” level ofexpression of a marker is the level of expression, copy number of themarker, or copy number of the MCR, in a biological sample, e.g., asample containing tissue, whole blood, serum, plasma, buccal scrape,saliva, cerebrospinal fluid, urine, stool, bronchoalveolar lavage, andlung tissue, from a subject, e.g., a human, not afflicted with cancer.

The term “altered level of expression” of a marker or MCR refers to anexpression level or copy number of a marker in a test sample e.g., asample derived from a patient suffering from cancer, that is greater orless than the standard error of the assay employed to assess expressionor copy number, and is preferably at least twice, and more preferablythree, four, five or ten or more times the expression level or copynumber of the marker or MCR in a control sample (e.g., sample from ahealthy subjects not having the associated disease) and preferably, theaverage expression level or copy number of the marker or MCR in severalcontrol samples. The altered level of expression is greater or less thanthe standard error of the assay employed to assess expression or copynumber, and is preferably at least twice, and more preferably three,four, five or ten or more times the expression level or copy number ofthe marker or MCR in a control sample (e.g., sample from a healthysubjects not having the associated disease) and preferably, the averageexpression level or copy number of the marker or MCR in several controlsamples.

An “overexpression” or “significantly higher level of expression or copynumber” of a marker or MCR refers to an expression level or copy numberin a test sample that is greater than the standard error of the assayemployed to assess expression or copy number, and is preferably at leasttwice, and more preferably three, four, five or ten or more times theexpression level or copy number of the marker or MCR in a control sample(e.g., sample from a healthy subject not afflicted with cancer) andpreferably, the average expression level or copy number of the marker orMCR in several control samples.

An “underexpression” or “significantly lower level of expression or copynumber” of a marker or MCR refers to an expression level or copy numberin a test sample that is greater than the standard error of the assayemployed to assess expression or copy number, but is preferably at leasttwice, and more preferably three, four, five or ten or more times lessthan the expression level or copy number of the marker or MCR in acontrol sample (e.g., sample from a healthy subject not afflicted withcancer) and preferably, the average expression level or copy number ofthe marker or MCR in several control samples.

“Methylation status” of a marker refers to the methylation pattern,e.g., methylation of the promoter of the marker, and/or methylationlevels of the marker. DNA methylation is a heritable, reversible andepigenetic change. Yet, DNA methylation has the potential to alter geneexpression, which has developmental and genetic consequences. DNAmethylation has been linked to cancer, as described in, for example,Laird, et al. (1994) Human Molecular Genetics 3:1487-1495 and Laird, P.(2003) Nature 3:253-266, the contents of which are incorporated hereinby reference. For example, methylation of CpG oligonucleotides in thepromoters of tumor suppressor genes can lead to their inactivation. Inaddition, alterations in the normal methylation process are associatedwith genomic instability (Lengauer. et al. Proc. Natl. Acad. Sci. USA94:2545-2550, 1997). Such abnormal epigenetic changes may be found inmany types of cancer and can, therefore, serve as potential markers foroncogenic transformation.

Methods for determining methylation include restriction landmark genomicscanning (Kawai, et al., Mol. Cell. Biol. 14:7421-7427, 1994),methylation-sensitive arbitrarily primed PCR (Gonzalgo, et al., CancerRes. 57:594-599, 1997); digestion of genomic DNA withmethylation-sensitive restriction enzymes followed by Southern analysisof the regions of interest (digestion-Southern method); PCR-basedprocess that involves digestion of genomic DNA withmethylation-sensitive restriction enzymes prior to PCR amplification(Singer-Sam, et al., Nucl. Acids Res. 18:687, 1990); genomic sequencingusing bisulfite treatment (Frommer, et al., Proc. Natl. Acad. Sci. USA89:1827-1831, 1992); methylation-specific PCR (NSP) (Herman, et al.Proc. Natl. Acad. Sci. USA 93:9821-9826, 1992); and restriction enzymedigestion of PCR products amplified from bisulfite-converted DNA (Sadriand Hornsby, Nucl. Acids Res. 24:5058-5059, 1996; and Xiong and Laird,Nucl. Acids. Res. 25:2532-2534, 1997); PCR techniques for detection ofgene mutations (Kuppuswamy, et al., Proc. Natl. Acad. Sci. USA88:1143-1147, 1991) and quantitation of allelic-specific expression(Szabo and Mann, Genes Dev. 9:3097-3108, 1995; and Singer-Sam, et al.,PCR Methods Appl. 1:160-163, 1992); and methods described in U.S. Pat.No. 6,251,594, the contents of which are incorporated herein byreference. An integrated genomic and epigenomic analysis as described inZardo, et al. (2000) Nature Genetics 32:453-458, may also be used.

The term “altered activity” of a marker refers to an activity of amarker which is increased or decreased in a disease state, e.g., in acancer sample, as compared to the activity of the marker in a normal,control sample. Altered activity of a marker may be the result of, forexample, altered expression of the marker, altered protein level of themarker, altered structure of the marker, or, e.g., an alteredinteraction with other proteins involved in the same or differentpathway as the marker or altered interaction with transcriptionalactivators or inhibitors, or altered methylation status.

The term “altered structure” of a marker refers to the presence ofmutations or allelic variants within the marker gene or maker protein,e.g., mutations which affect expression or activity of the marker, ascompared to the normal or wild-type gene or protein. For example,mutations include, but are not limited to substitutions, deletions, oraddition mutations. Mutations may be present in the coding or non-codingregion of the marker.

A “marker nucleic acid” is a nucleic acid (e.g., DNA, mRNA, cDNA)encoded by or corresponding to a marker of the invention. For example,such marker nucleic acid molecules include DNA (e.g., cDNA) comprisingthe entire or a partial sequence of any of the nucleic acid sequencesset forth in Tables 1 or 2 or the complement or hybridizing fragment ofsuch a sequence. The marker nucleic acid molecules also include RNAcomprising the entire or a partial sequence of any of the nucleic acidsequences set forth in Tables 1 or 2 or the complement of such asequence, wherein all thymidine residues are replaced with uridineresidues. A “marker protein” is a protein encoded by or corresponding toa marker of the invention. A marker protein comprises the entire or apartial sequence of a protein encoded by any of the sequences set forthin Tables 1 or 2 or a fragment thereof. The terms “protein” and“polypeptide” are used interchangeably herein.

A “marker,” as used herein, includes any nucleic acid sequence presentin an MCR as set forth in Tables 3 and/or 7, or a protein encoded bysuch a sequence.

Markers identified herein include diagnostic and therapeutic markers. Asingle marker may be a diagnostic marker, a therapeutic marker, or botha diagnostic and therapeutic marker.

As used herein, the term “therapeutic marker” includes markers, e.g.,markers set forth in Tables 1 and 2, which are believed to be involvedin the development (including maintenance, progression, angiogenesis,and/or metastasis) of cancer. The cancer-related functions of atherapeutic marker may be confirmed by, e.g., (1) increased or decreasedcopy number (by, e.g., fluorescence in situ hybridization (FISH), andFISH plus spectral karotype (SKY), or quantitative PCR (qPCR)) ormutation (e.g., by sequencing), overexpression or underexpression (e.g.,by in situ hybridization (ISH), Northern Blot, or qPCR), increased ordecreased protein levels (e.g., by immunohistochemistry (IHC)), orincreased or decreased protein activity (determined by, for example,modulation of a pathway in which the marker is involved), e.g., in morethan about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,or more of human cancers; (2) the inhibition of cancer cellproliferation and growth, e.g., in soft agar, by, e.g., RNA interference(“RNAi”) of the marker; (3) the ability of the marker to enhancetransformation of mouse embryo fibroblasts (MEFs) by oncogenes, e.g.,Myc and RAS, or by RAS alone; (4) the ability of the marker to enhanceor decrease the growth of tumor cell lines, e.g., in soft agar; (5) theability of the marker to transform primary mouse cells in SCID explant;and/or; (6) the prevention of maintenance or formation of tumors, e.g.,tumors arising de novo in an animal or tumors derived from human cancercell lines, by inhibiting or activating the marker. In one embodiment, atherapeutic marker may be used as a diagnostic marker.

As used herein, the term “diagnostic marker” includes markers, e.g.,markers set forth in Tables 1 and 2, which are useful in the diagnosisof cancer, e.g., over- or under-activity emergence, expression, growth,remission, recurrence or resistance of tumors before, during or aftertherapy. The predictive functions of the marker may be confirmed by,e.g., (1) increased or decreased copy number (e.g., by FISH, FISH plusSKY, or qPCR), overexpression or underexpression (e.g., by ISH, NorthernBlot, or qPCR), increased or decreased protein level (e.g., by IHC), orincreased or decreased activity (determined by, for example, modulationof a pathway in which the marker is involved), e.g., in more than about5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, or more ofhuman cancers; (2) its presence or absence in a biological sample, e.g.,a sample containing tissue, whole blood, serum, plasma, buccal scrape,saliva, cerebrospinal fluid, urine, stool, bronchoalveolar lavage,and/or lung tissue from a subject, e.g. a human, afflicted with cancer;(3) its presence or absence in clinical subset of patients with cancer(e.g., those responding to a particular therapy or those developingresistance).

Diagnostic markers also include “surrogate markers,” e.g., markers whichare indirect markers of cancer progression.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example a markerof the invention. Probes can be either synthesized by one skilled in theart, or derived from appropriate biological preparations. For purposesof detection of the target molecule, probes may be specifically designedto be labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic monomers.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a spatially or temporally restricted manner.

An “RNA interfering agent” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target gene, e.g., a markerof the invention, by RNA interference (RNAi). Such RNA interferingagents include, but are not limited to, nucleic acid molecules includingRNA molecules which are homologous to the target gene, e.g., a marker ofthe invention, or a fragment thereof, short interfering RNA (siRNA), andsmall molecules which interfere with or inhibit expression of a targetgene by RNA interference (RNAi).

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G.and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibitingexpression of the target gene. In one embodiment, the RNA is doublestranded RNA (dsRNA). This process has been described in plants,invertebrates, and mammalian cells. In nature, RNAi is initiated by thedsRNA-specific endonuclease Dicer, which promotes processive cleavage oflong dsRNA into double-stranded fragments termed siRNAs. siRNAs areincorporated into a protein complex that recognizes and cleaves targetmRNAs. RNAi can also be initiated by introducing nucleic acid molecules,e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silencethe expression of target genes. As used herein, “inhibition of targetgene expression” or “inhibition of marker gene expression” includes anydecrease in expression or protein activity or level of the target gene(e.g., a marker gene of the invention) or protein encoded by the targetgene, e.g., a marker protein of the invention. The decrease may be of atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as comparedto the expression of a target gene or the activity or level of theprotein encoded by a target gene which has not been targeted by an RNAinterfering agent.

“Short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as an agent which functions to inhibitexpression of a target gene, e.g., by RNAi. An siRNA may be chemicallysynthesized, may be produced by in vitro transcription, or may beproduced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the over hang on one strand is notdependent on the length of the overhang on the second strand. Preferablythe siRNA is capable of promoting RNA interference through degradationor specific post-transcriptional gene silencing (PTGS) of the targetmessenger RNA (mRNA).

In another embodiment, an siRNA is a small hairpin (also called stemloop) RNA (shRNA). In one embodiment, these shRNAs are composed of ashort (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501 incorporated be reference herein).

RNA interfering agents, e.g., siRNA molecules, may be administered to apatient having or at risk for having cancer, to inhibit expression of amarker gene of the invention, e.g., a marker gene which is overexpressedin cancer (such as the markers listed in Table 2) and thereby treat,prevent, or inhibit cancer in the subject.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “transcribed polynucleotide” is a polynucleotide (e.g. an RNA, a cDNA,or an analog of one of an RNA or cDNA) which is complementary to orhomologous with all or a portion of a mature RNA made by transcriptionof a marker of the invention and normal post-transcriptional processing(e.g. splicing), if any, of the transcript, and reverse transcription ofthe transcript.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence similarity between two polynucleotide sequences orbetween two polypeptide sequences, with identity being a more strictcomparison. The phrases “percent identity or homology” and “% identityor homology” refer to the percentage of sequence similarity found in acomparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value there between.Identity or similarity can be determined by comparing a position in eachsequence that may be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical or matching nucleotides at positions sharedby the polynucleotide sequences. A degree of identity of polypeptidesequences is a function of the number of identical amino acids atpositions shared by the polypeptide sequences. A degree of homology orsimilarity of polypeptide sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences. The term“substantial homology,” as used herein, refers to homology of at least50%, more preferably, 60%, 70%, 80%, 90%, 95% or more.

A marker is “fixed” to a substrate if it is covalently or non-covalentlyassociated with the substrate such the substrate can be rinsed with afluid (e.g. standard saline citrate, pH 7.4) without a substantialfraction of the marker dissociating from the substrate.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g. encodes a natural protein).

Cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cancer isalso “inhibited” if recurrence or metastasis of the cancer is reduced,slowed, delayed, or prevented.

A kit is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g. a probe, for specifically detecting a marker ofthe invention, the manufacture being promoted, distributed, or sold as aunit for performing the methods of the present invention.

II. USES OF THE INVENTION

The present invention is based, in part, on the identification ofchromosomal regions (MCRs) which are structurally altered leading to adifferent copy number in cancer cells as compared to normal (i.e.non-cancerous) cells. Furthermore, the present invention is based, inpart, on the identification of markers, e.g., markers which reside inthe MCRs of the invention, which have an altered amount, structure,and/or activity in cancer cells as compared to normal (i.e.,non-cancerous) cells. The markers of the invention correspond to DNA,cDNA, RNA, and polypeptide molecules which can be detected in one orboth of normal and cancerous cells.

The amount, structure, and/or activity, e.g., the presence, absence,copy number, expression level, protein level, protein activity, presenceof mutations, e.g., mutations which affect activity of the marker (e.g.,substitution, deletion, or addition mutations), and/or methylationstatus, of one or more of these markers in a sample, e.g., a samplecontaining tissue, whole blood, serum, plasma, buccal scrape, saliva,cerebrospinal fluid, urine, stool, bronchoalveolar lavage, and lungtissue, is herein correlated with the cancerous state of the tissue. Inaddition, the presence, absence, and/or copy number of one or more ofthe MCRs of the invention in a sample is also correlated with thecancerous state of the tissue. The invention thus provides compositions,kits, and methods for assessing the cancerous state of cells (e.g. cellsobtained from a non-human, cultured non-human cells, and in vivo cells)as well as methods for treatment, prevention, and/or inhibition ofcancer using a modulator, e.g., an agonist or antagonist, of a marker ofthe invention.

The compositions, kits, and methods of the invention have the followinguses, among others:

1) assessing whether a subject is afflicted with cancer;

2) assessing the stage of cancer in a human subject;

3) assessing the grade of cancer in a subject;

4) assessing the benign or malignant nature of cancer in a subject;

5) assessing the metastatic potential of cancer in a subject;

6) assessing the histological type of neoplasm associated with cancer ina subject;

7) making antibodies, antibody fragments or antibody derivatives thatare useful for treating cancer and/or assessing whether a subject isafflicted with cancer;

8) assessing the presence of cancer cells;

9) assessing the efficacy of one or more test compounds for inhibitingcancer in a subject;

10) assessing the efficacy of a therapy for inhibiting cancer in asubject;

11) monitoring the progression of cancer in a subject;

12) selecting a composition or therapy for inhibiting cancer, e.g., in asubject;

13) treating a subject afflicted with cancer;

14) inhibiting cancer in a subject;

15) assessing the carcinogenic potential of a test compound; and

16) preventing the onset of cancer in a subject at risk for developingcancer.

The invention thus includes a method of assessing whether a subject isafflicted with cancer or is at risk for developing cancer. This methodcomprises comparing the amount, structure, and/or activity, e.g., thepresence, absence, copy number, expression level, protein level, proteinactivity, presence of mutations, e.g., mutations which affect activityof the marker (e.g., substitution, deletion, or addition mutations),and/or methylation status, of a marker in a subject sample with thenormal level. A significant difference between the amount, structure, oractivity of the marker in the subject sample and the normal level is anindication that the subject is afflicted with cancer. The invention alsoprovides a method for assessing whether a subject is afflicted withcancer or is at risk for developing cancer by comparing the level ofexpression of marker(s) within an MCR or copy number of an MCR in acancer sample with the level of expression of marker(s) within an MCR orcopy number of an MCR in a normal, control sample. A significantdifference between the level of expression of marker(s) within an MCR orcopy number of the MCR in the subject sample and the normal level is anindication that the subject is afflicted with cancer. The MCR isselected from the group consisting of those listed in Tables 3 and/or 7.

The marker is selected from the group consisting of the markers listedin Tables 1 and 2. Table 1 lists markers which have a highly significantcorrelation between gene expression and gene dosage (p, 0.05). The levelof expression or copy number of these markers is decreased in sampleshistologically identified as lung cancer, e.g., NSCLC. Table 1 alsolists the chromosome, physical position, in Mb, and Locus ID No:, foreach of the markers. Although one or more molecules corresponding to themarkers listed in Table 1 may have been described by others, thesignificance of these markers with regard to the cancerous state ofcells, has not previously been identified.

Table 2 also lists markers which have a highly significant correlationbetween gene expression and gene dosage (p, 0.05). The level ofexpression or copy number of these markers is increased in sampleshistologically identified as lung cancer, e.g., NSCLC. Table 2 alsolists the chromosome, physical position, in Mb, and Locus ID No:, foreach of the markers. Although one or more molecules corresponding to themarkers listed in Table 2 may have been described by others, thesignificance of these markers with regard to the cancerous state ofcells, has not previously been identified.

Any marker or combination of markers listed in Tables 1 or 2 or any MCRor combination of MCRs listed in Tables 3 and/or 7, may be used in thecompositions, kits, and methods of the present invention. In general, itis preferable to use markers for which the difference between theamount, e.g., level of expression or copy number, and/or activity of themarker or MCR in cancer cells and the amount, e.g., level of expressionor copy number, and/or activity of the same marker in normal cells, isas great as possible. Although this difference can be as small as thelimit of detection of the method for assessing amount and/or activity ofthe marker, it is preferred that the difference be at least greater thanthe standard error of the assessment method, and preferably a differenceof at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 100-,500-, 1000-fold or greater than the amount, e.g., level of expression orcopy number, and/or activity of the same biomarker in normal tissue.

It is understood that by routine screening of additional subject samplesusing one or more of the markers of the invention, it will be realizedthat certain of the markers have altered amount, structure, and/oractivity in cancers of various types, including specific lung cancers,e.g., NSCLC, as well as other cancers, examples of which include, butare not limited to, melanomas, breast cancer, bronchus cancer,colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer,ovarian cancer, urinary bladder cancer, brain or central nervous systemcancer, peripheral nervous system cancer, esophageal cancer, cervicalcancer, uterine or endometrial cancer, cancer of the oral cavity orpharynx, liver cancer, kidney cancer, testicular cancer, biliary tractcancer, small bowel or appendix cancer, salivary gland cancer, thyroidgland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancerof hematological tissues, and the like.

For example, it will be confirmed that some of the markers of theinvention have altered amount, structure, and/or activity in some, i.e.,10%, 20%, 30%, or 40%, or most (i.e. 50% or more) or substantially all(i.e. 80% or more) of cancer, e.g., lung cancer. Furthermore, it will beconfirmed that certain of the markers of the invention are associatedwith cancer of various histologic subtypes.

In addition, as a greater number of subject samples are assessed foraltered amount, structure, and/or activity of the markers or alteredexpression or copy number MCRs of the invention and the outcomes of theindividual subjects from whom the samples were obtained are correlated,it will also be confirmed that markers have altered amount, structure,and/or activity of certain of the markers or altered expression or copynumber of MCRs of the invention are strongly correlated with malignantcancers and that altered expression of other markers of the inventionare strongly correlated with benign tumors or premalignant states. Thecompositions, kits, and methods of the invention are thus useful forcharacterizing one or more of the stage, grade, histological type, andbenign/premalignant/malignant nature of cancer in subjects.

When the compositions, kits, and methods of the invention are used forcharacterizing one or more of the stage, grade, histological type, andbenign/premalignant/malignant nature of cancer, in a subject, it ispreferred that the marker or MCR or panel of markers or MCRs of theinvention be selected such that a positive result is obtained in atleast about 20%, and preferably at least about 40%, 60%, or 80%, andmore preferably, in substantially all, subjects afflicted with cancer,of the corresponding stage, grade, histological type, orbenign/premalignant/malignant nature. Preferably, the marker or panel ofmarkers of the invention is selected such that a PPV (positivepredictive value) of greater than about 10% is obtained for the generalpopulation (more preferably coupled with an assay specificity greaterthan 99.5%).

When a plurality of markers or MCRs of the invention are used in thecompositions, kits, and methods of the invention, the amount, structure,and/or activity of each marker or level of expression or copy number canbe compared with the normal amount, structure, and/or activity of eachof the plurality of markers or level of expression or copy number, innon-cancerous samples of the same type, either in a single reactionmixture (i.e., using reagents, such as different fluorescent probes, foreach marker) or in individual reaction mixtures corresponding to one ormore of the markers or MCRs.

In one embodiment, a significantly altered amount, structure, and/oractivity of more than one of the plurality of markers, or significantlyaltered copy number of one or more of the MCRs in the sample, relativeto the corresponding normal levels, is an indication that the subject isafflicted with cancer. For example, a significantly lower copy number inthe sample of each of the plurality of markers or MCRs, relative to thecorresponding normal levels or copy number, is an indication that thesubject is afflicted with cancer. In yet another embodiment, asignificantly enhanced copy number of one or more markers or MCRs and asignificantly lower level of expression or copy number of one or moremarkers or MCRs in a sample relative to the corresponding normal levels,is an indication that the subject is afflicted with cancer. Also, forexample, a significantly enhanced copy number in the sample of each ofthe plurality of markers or MCRs, relative to the corresponding normalcopy number, is an indication that the subject is afflicted with cancer.In yet another embodiment, a significantly enhanced copy number of oneor more markers or MCRs and a significantly lower copy number of one ormore markers or MCRs in a sample relative to the corresponding normallevels, is an indication that the subject is afflicted with cancer.

When a plurality of markers or MCRs are used, it is preferred that 2, 3,4, 5, 8, 10, 12, 15, 20, 30, or 50 or more individual markers or MCRs beused or identified, wherein fewer markers or MCRs are preferred.

Only a small number of markers are known to be associated with, forexample, lung cancer (e.g., MYC, p16^(INK4A), RB1, RASSF1, TUSC2,SEMA3B, FHIT, and KRAS2). These markers or other markers which are knownto be associated with other types of cancer may be used together withone or more markers of the invention in, for example, a panel ofmarkers. In addition, frequent gains have been mapped to 1q31, 3q25-27,5p13-14 and 8q23-24 and losses to 3p21, 8p22, 9p21-22, 13q22, and17p12-13 (Bjorkqvist, A. M., et al. (1998) Br J Cancer 77, 260-9; Luk,C., et al. (2001) Cancer Genet Cytogenet 125, 87-99; Pei, J., et al.(2001) Genes Chromosomes Cancer 31, 282-7; Petersen, I., et al. (1997)Cancer Res 57, 2331-5; Balsara, B. R. & Testa, J. R. (2002) Oncogene 21,6877-83) in lung cancer. In some instances, validated oncogenes andtumor suppressor genes residing within these loci have been identified,including MYC (8q24), p16^(INK4A) (9p21), RB1 (13q14), RASSF1 (3p21),TUSC2 (3p21), SEMA3B (3p21), FHIT (3p14), and KRAS2 (12p12). It is wellknown that certain types of genes, such as oncogenes, tumor suppressorgenes, growth factor-like genes, protease-like genes, and proteinkinase-like genes are often involved with development of cancers ofvarious types. Thus, among the markers of the invention, use of thosewhich correspond to proteins which resemble known proteins encoded byknown oncogenes and tumor suppressor genes, and those which correspondto proteins which resemble growth factors, proteases, and proteinkinases, are preferred.

It is recognized that the compositions, kits, and methods of theinvention will be of particular utility to subjects having an enhancedrisk of developing cancer, and their medical advisors. Subjectsrecognized as having an enhanced risk of developing cancer, include, forexample, subjects having a familial history of cancer, subjectsidentified as having a mutant oncogene (i.e. at least one allele), andsubjects of advancing age.

An alteration, e.g. copy number, amount, structure, and/or activity of amarker in normal (i.e. non-cancerous) human tissue can be assessed in avariety of ways. In one embodiment, the normal level of expression orcopy number is assessed by assessing the level of expression and/or copynumber of the marker or MCR in a portion of cells which appear to benon-cancerous and by comparing this normal level of expression or copynumber with the level of expression or copy number in a portion of thecells which are suspected of being cancerous. For example, whenlaparoscopy or other medical procedure, reveals the presence of a tumoron one portion of an organ, the normal level of expression or copynumber of a marker or MCR may be assessed using the non-affected portionof the organ, and this normal level of expression or copy number may becompared with the level of expression or copy number of the same markerin an affected portion (i.e., the tumor) of the organ. Alternately, andparticularly as further information becomes available as a result ofroutine performance of the methods described herein, population-averagevalues for “normal” copy number, amount, structure, and/or activity ofthe markers or MCRs of the invention may be used. In other embodiments,the “normal” copy number, amount, structure, and/or activity of a markeror MCR may be determined by assessing copy number, amount, structure,and/or activity of the marker or MCR in a subject sample obtained from anon-cancer-afflicted subject, from a subject sample obtained from asubject before the suspected onset of cancer in the subject, fromarchived subject samples, and the like.

The invention includes compositions, kits, and methods for assessing thepresence of cancer cells in a sample (e.g. an archived tissue sample ora sample obtained from a subject). These compositions, kits, and methodsare substantially the same as those described above, except that, wherenecessary, the compositions, kits, and methods are adapted for use withcertain types of samples. For example, when the sample is a parafinized,archived human tissue sample, it may be necessary to adjust the ratio ofcompounds in the compositions of the invention, in the kits of theinvention, or the methods used. Such methods are well known in the artand within the skill of the ordinary artisan.

The invention thus includes a kit for assessing the presence of cancercells (e.g. in a sample such as a subject sample). The kit may compriseone or more reagents capable of identifying a marker or MCR of theinvention, e.g., binding specifically with a nucleic acid or polypeptidecorresponding to a marker or MCR of the invention. Suitable reagents forbinding with a polypeptide corresponding to a marker of the inventioninclude antibodies, antibody derivatives, antibody fragments, and thelike. Suitable reagents for binding with a nucleic acid (e.g. a genomicDNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementarynucleic acids. For example, the nucleic acid reagents may includeoligonucleotides (labeled or non-labeled) fixed to a substrate, labeledoligonucleotides not bound with a substrate, pairs of PCR primers,molecular beacon probes, and the like.

The kit of the invention may optionally comprise additional componentsuseful for performing the methods of the invention. By way of example,the kit may comprise fluids (e.g., SSC buffer) suitable for annealingcomplementary nucleic acids or for binding an antibody with a proteinwith which it specifically binds, one or more sample compartments, aninstructional material which describes performance of a method of theinvention, a sample of normal cells, a sample of cancer cells, and thelike.

A kit of the invention may comprise a reagent useful for determiningprotein level or protein activity of a marker. In another embodiment, akit of the invention may comprise a reagent for determining methylationstatus of a marker, or may comprise a reagent for determining alterationof structure of a marker, e.g., the presence of a mutation.

The invention also includes a method of making an isolated hybridomawhich produces an antibody useful in methods and kits of the presentinvention. A protein corresponding to a marker of the invention may beisolated (e.g. by purification from a cell in which it is expressed orby transcription and translation of a nucleic acid encoding the proteinin vivo or in vitro using known methods) and a vertebrate, preferably amammal such as a mouse, rat, rabbit, or sheep, is immunized using theisolated protein. The vertebrate may optionally (and preferably) beimmunized at least one additional time with the isolated protein, sothat the vertebrate exhibits a robust immune response to the protein.Splenocytes are isolated from the immunized vertebrate and fused with animmortalized cell line to form hybridomas, using any of a variety ofmethods well known in the art. Hybridomas formed in this manner are thenscreened using standard methods to identify one or more hybridomas whichproduce an antibody which specifically binds with the protein. Theinvention also includes hybridomas made by this method and antibodiesmade using such hybridomas.

The invention also includes a method of assessing the efficacy of a testcompound for inhibiting cancer cells. As described above, differences inthe amount, structure, and/or activity of the markers of the invention,or level of expression or copy number of the MCRs of the invention,correlate with the cancerous state of cells. Although it is recognizedthat changes in the levels of amount, e.g., expression or copy number,structure, and/or activity of certain of the markers or expression orcopy number of the MCRs of the invention likely result from thecancerous state of cells, it is likewise recognized that changes in theamount may induce, maintain, and promote the cancerous state. Thus,compounds which inhibit cancer, in a subject may cause a change, e.g., achange in expression and/or activity of one or more of the markers ofthe invention to a level nearer the normal level for that marker (e.g.,the amount, e.g., expression, and/or activity for the marker innon-cancerous cells).

This method thus comprises comparing amount, e.g., expression, and/oractivity of a marker in a first cell sample and maintained in thepresence of the test compound and amount, e.g., expression, and/oractivity of the marker in a second cell sample and maintained in theabsence of the test compound. A significant increase in the amount,e.g., expression, and/or activity of a marker listed in Table 1 (e.g., amarker that was shown to be decreased in cancer), a significant decreasein the amount, e.g., expression, and/or activity of a marker listed inTable 2 (e.g., a marker that was shown to be increased in cancer), is anindication that the test compound inhibits cancer. The cell samples may,for example, be aliquots of a single sample of normal cells obtainedfrom a subject, pooled samples of normal cells obtained from a subject,cells of a normal cell lines, aliquots of a single sample of cancer,cells obtained from a subject, pooled samples of cancer, cells obtainedfrom a subject, cells of a cancer cell line, cells from an animal modelof cancer, or the like. In one embodiment, the samples are cancer cellsobtained from a subject and a plurality of compounds known to beeffective for inhibiting various cancers, are tested in order toidentify the compound which is likely to best inhibit the cancer in thesubject.

This method may likewise be used to assess the efficacy of a therapy,e.g., chemotherapy, radiation therapy, surgery, or any other therapeuticapproach useful for inhibiting cancer in a subject. In this method, theamount, e.g., expression, and/or activity of one or more markers of theinvention in a pair of samples (one subjected to the therapy, the othernot subjected to the therapy) is assessed. As with the method ofassessing the efficacy of test compounds, if the therapy induces asignificant decrease in the amount, e.g., expression, and/or activity ofa marker listed in Table 2 (e.g., a marker that was shown to beincreased in cancer), blocks induction of a marker listed in Table 2(e.g., a marker that was shown to be increased in cancer), or if thetherapy induces a significant enhancement of the amount, e.g.,expression, and/or activity of a marker listed in Table 1 (e.g., amarker that was shown to be decreased in cancer), then the therapy isefficacious for inhibiting cancer. As above, if samples from a selectedsubject are used in this method, then alternative therapies can beassessed in vitro in order to select a therapy most likely to beefficacious for inhibiting cancer in the subject.

This method may likewise be used to monitor the progression of cancer ina subject, wherein if a sample in a subject has a significant decreasein the amount, e.g., expression, and/or activity of a marker listed inTable 2 (e.g., a marker that was shown to be increased in cancer, orblocks induction of a marker listed in Table 2 (e.g., a marker that wasshown to be increased in cancer), or a significant enhancement of theamount, e.g., expression, and/or activity of a marker listed in Table 1(e.g., a marker that was shown to be decreased in cancer), during theprogression of cancer, e.g., at a first point in time and a subsequentpoint in time, then the cancer has improved. In yet another embodiment,between the first point in time and a subsequent point in time, thesubject has undergone treatment, e.g., chemotherapy, radiation therapy,surgery, or any other therapeutic approach useful for inhibiting cancer,has completed treatment, or is in remission.

As described herein, cancer in subjects is associated with an increasein amount, e.g., expression, and/or activity of one or more markerslisted in Table 2 (e.g., a marker that was shown to be increased incancer), and/or a decrease in amount, e.g., expression, and/or activityof one or more markers listed in Table 1 (e.g., a marker that was shownto be decreased in cancer). While, as discussed above, some of thesechanges in amount, e.g., expression, and/or activity number result fromoccurrence of the cancer, others of these changes induce, maintain, andpromote the cancerous state of cancer cells. Thus, cancer characterizedby an increase in the amount, e.g., expression, and/or activity of oneor more markers listed in Table 2 (e.g., a marker that was shown to beincreased in cancer), can be inhibited by inhibiting amount, e.g.,expression, and/or activity of those markers. Likewise, cancercharacterized by a decrease in the amount, e.g., expression, and/oractivity of one or more markers listed in Table 1 (e.g., a marker thatwas shown to be decreased in cancer), can be inhibited by enhancingamount, e.g., expression, and/or activity of those markers.

Amount and/or activity of a marker listed in Table 2 (e.g., a markerthat was shown to be increased in cancer), can be inhibited in a numberof ways generally known in the art. For example, an antisenseoligonucleotide can be provided to the cancer cells in order to inhibittranscription, translation, or both, of the marker(s). An RNAinterfering agent, e.g., an siRNA molecule, which is targeted to amarker listed in Table 2, can be provided to the cancer cells in orderto inhibit expression of the target marker, e.g., through degradation orspecific post-transcriptional gene silencing (PTGS) of the messenger RNA(mRNA) of the target marker. Alternately, a polynucleotide encoding anantibody, an antibody derivative, or an antibody fragment, e.g., afragment capable of binding an antigen, and operably linked with anappropriate promoter or regulator region, can be provided to the cell inorder to generate intracellular antibodies which will inhibit thefunction, amount, and/or activity of the protein corresponding to themarker(s). Conjugated antibodies or fragments thereof, e.g.,chemolabeled antibodies, radiolabeled antibodies, or immunotoxinstargeting a marker of the invention may also be administered to treat,prevent or inhibit cancer.

A small molecule may also be used to modulate, e.g., inhibit, expressionand/or activity of a marker listed in Table 2. In one embodiment, asmall molecule functions to disrupt a protein-protein interactionbetween a marker of the invention and a target molecule or ligand,thereby modulating, e.g., increasing or decreasing the activity of themarker.

Using the methods described herein, a variety of molecules, particularlyincluding molecules sufficiently small that they are able to cross thecell membrane, can be screened in order to identify molecules whichinhibit amount and/or activity of the marker(s). The compound soidentified can be provided to the subject in order to inhibit amountand/or activity of the marker(s) in the cancer cells of the subject.

Amount and/or activity of a marker listed in Table 1 (e.g., a markerthat was shown to be decreased in cancer), can be enhanced in a numberof ways generally known in the art. For example, a polynucleotideencoding the marker and operably linked with an appropriatepromoter/regulator region can be provided to cells of the subject inorder to induce enhanced expression and/or activity of the protein (andmRNA) corresponding to the marker therein. Alternatively, if the proteinis capable of crossing the cell membrane, inserting itself in the cellmembrane, or is normally a secreted protein, then amount and/or activityof the protein can be enhanced by providing the protein (e.g. directlyor by way of the bloodstream) to cancer cells in the subject. A smallmolecule may also be used to modulate, e.g., increase, expression oractivity of a marker listed in Table 1. Furthermore, in anotherembodiment, a modulator of a marker of the invention, e.g., a smallmolecule, may be used, for example, to re-express a silenced gene, e.g.,a tumor suppressor, in order to treat or prevent cancer. For example,such a modulator may interfere with a DNA binding element or amethyltransferase.

As described above, the cancerous state of human cells is correlatedwith changes in the amount and/or activity of the markers of theinvention. Thus, compounds which induce increased expression or activityof one or more of the markers listed in Table 2 (e.g., a marker that wasshown to be increased in cancer), decreased amount and/or activity ofone or more of the markers listed in Table 1 (e.g., a marker that wasshown to be decreased in cancer), can induce cell carcinogenesis. Theinvention also includes a method for assessing the human cellcarcinogenic potential of a test compound. This method comprisesmaintaining separate aliquots of human cells in the presence and absenceof the test compound. Expression or activity of a marker of theinvention in each of the aliquots is compared. A significant increase inthe amount and/or activity of a marker listed in Table 2 (e.g., a markerthat was shown to be increased in cancer), or a significant decrease inthe amount and/or activity of a marker listed in Table 1 (e.g., a markerthat was shown to be decreased in cancer), in the aliquot maintained inthe presence of the test compound (relative to the aliquot maintained inthe absence of the test compound) is an indication that the testcompound possesses human cell carcinogenic potential. The relativecarcinogenic potentials of various test compounds can be assessed bycomparing the degree of enhancement or inhibition of the amount and/oractivity of the relevant markers, by comparing the number of markers forwhich the amount and/or activity is enhanced or inhibited, or bycomparing both.

III. ISOLATED NUCLEIC ACID MOLECULES

One aspect of the invention pertains to isolated nucleic acid moleculesthat correspond to a marker of the invention, including nucleic acidswhich encode a polypeptide corresponding to a marker of the invention ora portion of such a polypeptide. The nucleic acid molecules of theinvention include those nucleic acid molecules which reside in the MCRsidentified herein. Isolated nucleic acid molecules of the invention alsoinclude nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules that correspond to a marker ofthe invention, including nucleic acid molecules which encode apolypeptide corresponding to a marker of the invention, and fragments ofsuch nucleic acid molecules, e.g., those suitable for use as PCR primersfor the amplification or mutation of nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Preferably, an “isolated” nucleic acid moleculeis free of sequences (preferably protein-encoding sequences) whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kB, 4kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecules encoding a protein corresponding to a marker listed in Tables1 or 2, can be isolated using standard molecular biology techniques andthe sequence information in the database records described herein. Usingall or a portion of such nucleic acid sequences, nucleic acid moleculesof the invention can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook et al., ed.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid molecules so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which has a nucleotidesequence complementary to the nucleotide sequence of a nucleic acidcorresponding to a marker of the invention or to the nucleotide sequenceof a nucleic acid encoding a protein which corresponds to a marker ofthe invention. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention call comprise only aportion of a nucleic acid sequence, wherein the full length nucleic acidsequence comprises a marker of the invention or which encodes apolypeptide corresponding to a marker of the invention. Such nucleicacid molecules can be used, for example, as a probe or primer. Theprobe/primer typically is used as one or more substantially purifiedoligonucleotides. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 7, preferably about 15, more preferably about 25, 50, 75,100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutivenucleotides of a nucleic acid of the invention.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences corresponding toone or more markers of the invention. The probe comprises a label groupattached thereto, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as part of adiagnostic test kit for identifying cells or tissues which mis-expressthe protein, such as by measuring levels of a nucleic acid moleculeencoding the protein in a sample of cells from a subject, e.g.,detecting mRNA levels or determining whether a gene encoding the proteinhas been mutated or deleted.

The invention further encompasses nucleic acid molecules that differ,due to degeneracy of the genetic code, from the nucleotide sequence ofnucleic acid molecules encoding a protein which corresponds to a markerof the invention, and thus encode the same protein.

In addition to the nucleotide sequences described in Tables 1 or 2, itwill be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequence can existwithin a population (e.g., the human population). Such geneticpolymorphisms can exist among individuals within a population due tonatural allelic variation. An allele is one of a group of genes whichoccur alternatively at a given genetic locus. In addition, it will beappreciated that DNA polymorphisms that affect RNA expression levels canalso exist that may affect the overall expression level of that gene(e.g., by affecting regulation or degradation).

The term “allele,” which is used interchangeably herein with “allelicvariant,” refers to alternative forms of a gene or portions thereof.Alleles occupy the same locus or position on homologous chromosomes.When a subject has two identical alleles of a gene, the subject is saidto be homozygous for the gene or allele. When a subject has twodifferent alleles of a gene, the subject is said to be heterozygous forthe gene or allele. Alleles of a specific gene, including, but notlimited to, the genes listed in Tables 1, 2, or 3, can differ from eachother in a single nucleotide, or several nucleotides, and can includesubstitutions, deletions, and insertions of nucleotides. An allele of agene can also be a form of a gene containing one or more mutations.

The term “allelic variant of a polymorphic region of gene” or “allelicvariant”, used interchangeably herein, refers to an alternative form ofa gene having one of several possible nucleotide sequences found in thatregion of the gene in the population. As used herein, allelic variant ismeant to encompass functional allelic variants, non-functional allelicvariants, SNPs, mutations and polymorphisms.

The term “single nucleotide polymorphism” (SNP) refers to a polymorphicsite occupied by a single nucleotide, which is the site of variationbetween allelic sequences. The site is usually preceded by and followedby highly conserved sequences of the allele (e.g., sequences that varyin less than 1/100 or 1/1000 members of a population). A SNP usuallyarises due to substitution of one nucleotide for another at thepolymorphic site. SNPs can also arise from a deletion of a nucleotide oran insertion of a nucleotide relative to a reference allele. Typicallythe polymorphic site is occupied by a base other than the referencebase. For example, where the reference allele contains the base “T”(thymidine) at the polymorphic site, the altered allele can contain a“C” (cytidine), “G” (guanine), or “A” (adenine) at the polymorphic site.SNP's may occur in protein-coding nucleic acid sequences, in which casethey may give rise to a defective or otherwise variant protein, orgenetic disease. Such a SNP may alter the coding sequence of the geneand therefore specify another amino acid (a “missense” SNP) or a SNP mayintroduce a stop codon (a “nonsense” SNP). When a SNP does not alter theamino acid sequence of a protein, the SNP is called “silent.” SNP's mayalso occur in noncoding regions of the nucleotide sequence. This mayresult in defective protein expression, e.g., as a result of alternativespicing, or it may have no effect on the function of the protein.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptidecorresponding to a marker of the invention. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention.

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600,1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or morenucleotides in length and hybridizes under stringent conditions to anucleic acid molecule corresponding to a marker of the invention or to anucleic acid molecule encoding a protein corresponding to a marker ofthe invention. As used herein, the term “hybridizes under stringentconditions” is intended to describe conditions for hybridization andwashing under which nucleotide sequences at least 60% (65%, 70%, 75%,80%, preferably 85%) identical to each other typically remain hybridizedto each other. Such stringent conditions are known to those skilled inthe art and can be found in sections 6.3.1-6.3.6 of Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989). A preferred,non-limiting example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention that can exist in the population, the skilledartisan will further appreciate that sequence changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded protein, without altering the biological activity of the proteinencoded thereby. For example, one can make nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, amino acid residues that are notconserved or only semi-conserved among homologs of various species maybe non-essential for activity and thus would be likely targets foralteration. Alternatively, amino acid residues that are conserved amongthe homologs of various species (e.g., murine and human) may beessential for activity and thus would not be likely targets foralteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from the naturally-occurringproteins which correspond to the markers of the invention, yet retainbiological activity. In one embodiment, such a protein has an amino acidsequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%,95%, or 98% identical to the amino acid sequence of one of the proteinswhich correspond to the markers of the invention.

An isolated nucleic acid molecule encoding a variant protein can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of nucleic acids of theinvention, such that one or more amino acid residue substitutions,additions, or deletions are introduced into the encoded protein.Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid of theinvention, e.g., complementary to the coding strand of a double-strandedcDNA molecule corresponding to a marker of the invention orcomplementary to an mRNA sequence corresponding to a marker of theinvention. Accordingly, an antisense nucleic acid molecule of theinvention can hydrogen bond to (i.e. anneal with) a sense nucleic acidof the invention. The antisense nucleic acid can be complementary to anentire coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleicacid molecule can also be antisense to all or part of a non-codingregion of the coding strand of a nucleotide sequence encoding apolypeptide of the invention. The non-coding regions (“5′ and 3′untranslated regions”) are the 5′ and 3′ sequences which flank thecoding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been sub-cloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a polypeptidecorresponding to a selected marker of the invention to thereby inhibitexpression of the marker, e.g., by inhibiting transcription and/ortranslation. The hybridization can be by conventional nucleotidecomplementarity to form a stable duplex, or, for example, in the case ofan antisense nucleic acid molecule which binds to DNA duplexes, throughspecific interactions in the major groove of the double helix. Examplesof a route of administration of antisense nucleic acid molecules of theinvention includes direct injection at a tissue site or infusion of theantisense nucleic acid into a lung-associated body fluid. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes asdescribed in Haselhoff and Gerlach, 1988, Nature 334:585-591) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptidecorresponding to a marker of the invention can be designed based uponthe nucleotide sequence of a cDNA corresponding to the marker. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved (see Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, anmRNA encoding a polypeptide of the invention can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules (see, e.g., Bartel and Szostak, 1993, Science 261:1411-1418).

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, expression of a polypeptide of theinvention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In various embodiments, the nucleic acid molecules of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacid molecules can be modified to generate peptide nucleic acidmolecules (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs”refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996)Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc.Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNASE H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The invention also includes molecular beacon nucleic acid moleculeshaving at least one region which is complementary to a nucleic acidmolecule of the invention, such that the molecular beacon is useful forquantitating the presence of the nucleic acid molecule of the inventionin a sample. A “molecular beacon” nucleic acid is a nucleic acidmolecule comprising a pair of complementary regions and having afluorophore and a fluorescent quencher associated therewith. Thefluorophore and quencher are associated with different portions of thenucleic acid in such an orientation that when the complementary regionsare annealed with one another, fluorescence of the fluorophore isquenched by the quencher. When the complementary regions of the nucleicacid molecules are not annealed with one another, fluorescence of thefluorophore is quenched to a lesser degree. Molecular beacon nucleicacid molecules are described, for example, in U.S. Pat. No. 5,876,930.

IV. ISOLATED PROTEINS AND ANTIBODIES

One aspect of the invention pertains to isolated proteins whichcorrespond to individual markers of the invention, and biologicallyactive portions thereof, as well as polypeptide fragments suitable foruse as immunogens to raise antibodies directed against a polypeptidecorresponding to a marker of the invention. In one embodiment, thenative polypeptide corresponding to a marker can be isolated from cellsor tissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, polypeptidescorresponding to a marker of the invention are produced by recombinantDNA techniques. Alternative to recombinant expression, a polypeptidecorresponding to a marker of the invention can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide corresponding to a markerof the invention include polypeptides comprising amino acid sequencessufficiently identical to or derived from the amino acid sequence of theprotein corresponding to the marker (e.g., the protein encoded by thenucleic acid molecules listed in Tables 1 or 2), which include feweramino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding protein. A biologically active portionof a protein of the invention can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of the native form of a polypeptideof the invention.

Preferred polypeptides have an amino acid sequence of a protein encodedby a nucleic acid molecule listed in Tables 1 or 2. Other usefulproteins are substantially identical (e.g., at least about 40%,preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of thesesequences and retain the functional activity of the protein of thecorresponding naturally-occurring protein yet differ in amino acidsequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Suchan algorithm is incorporated into the ALIGN program (version 2.0) whichis part of the GCG sequence alignment software package. When utilizingthe ALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. Yet another useful algorithm for identifying regions of localsequence similarity and alignment is the FASTA algorithm as described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. Whenusing the FASTA algorithm for comparing nucleotide or amino acidsequences, a PAM120 weight residue table can, for example, be used witha k-tuple value of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides chimeric or fusion proteins corresponding toa marker of the invention. As used herein, a “chimeric protein” or“fusion protein” comprises all or part (preferably a biologically activepart) of a polypeptide corresponding to a marker of the inventionoperably linked to a heterologous polypeptide (i.e., a polypeptide otherthan the polypeptide corresponding to the marker). Within the fusionprotein, the term “operably linked” is intended to indicate that thepolypeptide of the invention and the heterologous polypeptide are fusedin-frame to each other. The heterologous polypeptide can be fused to theamino-terminus or the carboxyl-terminus of the polypeptide of theinvention.

One useful fusion protein is a GST fusion protein in which a polypeptidecorresponding to a marker of the invention is fused to the carboxylterminus of GST sequences. Such fusion proteins can facilitate thepurification of a recombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signalsequence at its amino terminus. For example, the native signal sequenceof a polypeptide corresponding to a marker of the invention can beremoved and replaced with a signal sequence from another protein. Forexample, the gp67 secretory sequence of the baculovirus envelope proteincan be used as a heterologous signal sequence (Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1992).Other examples of eukaryotic heterologous signal sequences include thesecretory sequences of melittin and human placental alkaline phosphatase(Stratagene; La Jolla, Calif.). In yet another example, usefulprokaryotic heterologous signal sequences include the phoA secretorysignal (Sambrook et al., supra) and the protein A secretory signal(Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an immunoglobulinfusion protein in which all or part of a polypeptide corresponding to amarker of the invention is fused to sequences derived from a member ofthe immunoglobulin protein family. The immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions andadministered to a subject to inhibit an interaction between a ligand(soluble or membrane-bound) and a protein on the surface of a cell(receptor), to thereby suppress signal transduction in vivo. Theimmunoglobulin fusion protein can be used to affect the bioavailabilityof a cognate ligand of a polypeptide of the invention. Inhibition ofligand/receptor interaction can be useful therapeutically, both fortreating proliferative and differentiative disorders and for modulating(e.g. promoting or inhibiting) cell survival. Moreover, theimmunoglobulin fusion proteins of the invention can be used asimmunogens to produce antibodies directed against a polypeptide of theinvention in a subject, to purify ligands and in screening assays toidentify molecules which inhibit the interaction of receptors withligands.

Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

A signal sequence can be used to facilitate secretion and isolation ofthe secreted protein or other proteins of interest. Signal sequences aretypically characterized by a core of hydrophobic amino acids which aregenerally cleaved from the mature protein during secretion in one ormore cleavage events. Such signal peptides contain processing sites thatallow cleavage of the signal sequence from the mature proteins as theypass through the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as topolypeptides from which the signal sequence has been proteolyticallycleaved (i.e., the cleavage products). In one embodiment, a nucleic acidsequence encoding a signal sequence can be operably linked in anexpression vector to a protein of interest, such as a protein which isordinarily not secreted or is otherwise difficult to isolate. The signalsequence directs secretion of the protein, such as from a eukaryotichost into which the expression vector is transformed, and the signalsequence is subsequently or concurrently cleaved. The protein can thenbe readily purified from the extracellular medium by art recognizedmethods. Alternatively, the signal sequence can be linked to the proteinof interest using a sequence which facilitates purification, such aswith a GST domain.

The present invention also pertains to variants of the polypeptidescorresponding to individual markers of the invention. Such variants havean altered amino acid sequence which can function as either agonists(mimetics) or as antagonists. Variants can be generated by mutagenesis,e.g., discrete point mutation or truncation. An agonist can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the protein. An antagonist of a protein caninhibit one or more of the activities of the naturally occurring form ofthe protein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade which includes theprotein of interest. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theprotein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the inventionfrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang,1983, Tetrahedron 39:3; Itakura et al, 1984, Annu. Rev. Biochem. 53:323;Itakura et al., 1984, Science 198:1056; Ike et al., 1983 Nucleic AcidRes. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide corresponding to a marker of the invention can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variants. For example, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of the coding sequence of interest with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes amino terminal andinternal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high throughputanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al, 1993, Protein Engineering6(3):327-331).

An isolated polypeptide corresponding to a marker of the invention, or afragment thereof, can be used as an immunogen to generate antibodiesusing standard techniques for polyclonal and monoclonal antibodypreparation. The full-length polypeptide or protein can be used or,alternatively, the invention provides antigenic peptide fragments foruse as immunogens. The antigenic peptide of a protein of the inventioncomprises at least 8 (preferably 10, 15, 20, or 30 or more) amino acidresidues of the amino acid sequence of one of the polypeptides of theinvention, and encompasses an epitope of the protein such that anantibody raised against the peptide forms a specific immune complex witha marker of the invention to which the protein corresponds. Preferredepitopes encompassed by the antigenic peptide are regions that arelocated on the surface of the protein, e.g., hydrophilic regions.Hydrophobicity sequence analysis, hydrophilicity sequence analysis, orsimilar analyses can be used to identify hydrophilic regions.

An immunogen typically is used to prepare antibodies by immunizing asuitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse,or other mammal or vertebrate. An appropriate immunogenic preparationcan contain, for example, recombinantly-expressed orchemically-synthesized polypeptide. The preparation can further includean adjuvant, such as Freund's complete or incomplete adjuvant, or asimilar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodiesdirected against a polypeptide of the invention. The terms “antibody”and “antibody substance” as used interchangeably herein refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen, such as a polypeptideof the invention. A molecule which specifically binds to a givenpolypeptide of the invention is a molecule which binds the polypeptide,but does not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains the polypeptide. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies. The term “monoclonal antibody” or“monoclonal antibody composition”, as used herein, refers to apopulation of antibody molecules that contain only one species of anantigen binding site capable of immunoreacting with a particularepitope.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide of the invention as an immunogen.The antibody titer in the immunized subject can be monitored over timeby standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be harvested or isolated from the subject (e.g., from theblood or serum of the subject) and further purified by well-knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), theEBV-hybridoma technique (see Cole et al., pp. 77-96 In MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Current Protocols in Immunology, Coligan et al. ed., JohnWiley & Sons, New York, 1994). Hybridoma cells producing a monoclonalantibody of the invention are detected by screening the hybridomaculture supernatants for antibodies that bind the polypeptide ofinterest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody display library can be found in, forexample, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; PCTPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human subjects. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide corresponding to a marker of the invention. Monoclonalantibodies directed against the antigen can be obtained usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995) Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S.Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016;and U.S. Pat. No. 5,545,806. In addition, companies such as Abgenix,Inc. (Freemont, Calif.), can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1994, Bio/technology12:899-903).

An antibody, antibody derivative, or fragment thereof, whichspecifically binds a marker of the invention which is overexpressed incancer (e.g., a marker set forth in Table 2), may be used to inhibitactivity of a marker, e.g., a marker set forth in Table 2, and thereforemay be administered to a subject to treat, inhibit, or prevent cancer inthe subject. Furthermore, conjugated antibodies may also be used totreat, inhibit, or prevent cancer in a subject. Conjugated antibodies,preferably monoclonal antibodies, or fragments thereof, are antibodieswhich are joined to drugs, toxins, or radioactive atoms, and used asdelivery vehicles to deliver those substances directly to cancer cells.The antibody, e.g., an antibody which specifically binds a marker of theinvention (e.g., a marker listed in Table 2), is administered to asubject and binds the marker, thereby delivering the toxic substance tothe cancer cell, minimizing damage to normal cells in other parts of thebody.

Conjugated antibodies are also referred to as “tagged,” “labeled,” or“loaded.” Antibodies with chemotherapeutic agents attached are generallyreferred to as chemolabeled. Antibodies with radioactive particlesattached are referred to as radiolabeled, and this type of therapy isknown as radioimmunotherapy (RIT). Aside from being used to treatcancer, radiolabeled antibodies can also be used to detect areas ofcancer spread in the body. Antibodies attached to toxins are calledimmunotoxins.

Immunotoxins are made by attaching toxins (e.g., poisonous substancesfrom plants or bacteria) to monoclonal antibodies. Immunotoxins may beproduced by attaching monoclonal antibodies to bacterial toxins such asdiphtherial toxin (DT) or pseudomonal exotoxin (PE40), or to planttoxins such as ricin A or saporin.

An antibody directed against a polypeptide corresponding to a marker ofthe invention (e.g., a monoclonal antibody) can be used to isolate thepolypeptide by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, such an antibody can be used to detectthe marker (e.g., in a cellular lysate or cell supernatant) in order toevaluate the level and pattern of expression of the marker. Theantibodies can also be used diagnostically to monitor protein levels intissues or body fluids (e.g. in a lung-associated body fluid) as part ofa clinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

V. RECOMBINANT EXPRESSION VECTORS AND HOST CELLS

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidecorresponding to a marker of the invention (or a portion of such apolypeptide). As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, namely expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Methods in Enzymology: GeneExpression Technology vol. 185, Academic Press, San Diego, Calif.(1991). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide corresponding to a marker of the inventionin prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells{using baculovirus expression vectors}, yeast cells or mammalian cells).Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., p. 60-89, In Gene Expression Technology: Methods in Enzymologyvol. 185, Academic Press, San Diego, Calif., 1991). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3)or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene underthe transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, p. 119-128,In Gene Expression Technology: Methods in Enzymology vol. 185, AcademicPress, San Diego, Calif., 1990. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., 1992, Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840)and pMT2PC (Kaufman et al, 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al,1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988, Adv. Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al, 1985,Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss,1990, Science 249:374-379) and the α-fetoprotein promoter (Camper andTilghman, 1989, Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoding a polypeptide of the invention.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue-specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid, or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub et al., 1986, Trends in Genetics, Vol. 1(1).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce a polypeptide corresponding to amarker of the invention. Accordingly, the invention further providesmethods for producing a polypeptide corresponding to a marker of theinvention using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding a polypeptide of the inventionhas been introduced) in a suitable medium such that the marker isproduced. In another embodiment, the method further comprises isolatingthe marker polypeptide from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichsequences encoding a polypeptide corresponding to a marker of theinvention have been introduced. Such host cells can then be used tocreate non-human transgenic animals in which exogenous sequencesencoding a marker protein of the invention have been introduced intotheir genome or homologous recombinant animals in which endogenousgene(s) encoding a polypeptide corresponding to a marker of theinvention sequences have been altered. Such animals are useful forstudying the function and/or activity of the polypeptide correspondingto the marker, for identifying and/or evaluating modulators ofpolypeptide activity, as well as in pre-clinical testing of therapeuticsor diagnostic molecules, for marker discovery or evaluation, e.g.,therapeutic and diagnostic marker discovery or evaluation, or assurrogates of drug efficacy and specificity.

As used herein, a “transgenic animal” is a non-human animal, preferablya mammal, more preferably a rodent such as a rat or mouse, in which oneor more of the cells of the animal includes a transgene. Other examplesof transgenic animals include non-human primates, sheep, dogs, cows,goats, chickens, amphibians, etc. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, an“homologous recombinant animal” is a non-human animal, preferably amammal, more preferably a mouse, in which an endogenous gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.Transgenic animals also include inducible transgenic animals, such asthose described in, for example, Chan I. T., et al. (2004) J ClinInvest. 113(4):528-38 and Chin L. et al (1999) Nature 400(6743):468-72.

A transgenic animal of the invention can be created by introducing anucleic acid encoding a polypeptide corresponding to a marker of theinvention into the male pronuclei of a fertilized oocyte, e.g., bymicroinjection, retroviral infection, and allowing the oocyte to developin a pseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the polypeptide of the invention toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986. Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the transgene in its genome and/or expressionof mRNA encoding the transgene in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying thetransgene can further be bred to other transgenic animals carrying othertransgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a gene encoding a polypeptidecorresponding to a marker of the invention into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the gene. In a preferred embodiment, the vector isdesigned such that, upon homologous recombination, the endogenous geneis functionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousgene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous protein). In the homologousrecombination vector, the altered portion of the gene is flanked at its5′ and 3′ ends by additional nucleic acid of the gene to allow forhomologous recombination to occur between the exogenous gene carried bythe vector and an endogenous gene in an embryonic stem cell. Theadditional flanking nucleic acid sequences are of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see, e.g., Thomas and Capecchi, 1987, Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced gene has homologouslyrecombined with the endogenous gene are selected (see, e.g., Li et al.,1992, Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, Ed., IRL, Oxford, 1987, pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al,1991, Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

VI. METHODS OF TREATMENT

The present invention provides for both prophylactic and therapeuticmethods of treating a subject, e.g., a human, who has or is at risk of(or susceptible to) cancer, e.g., lung cancer, e.g., NSCLC. As usedherein, “treatment” of a subject includes the application oradministration of a therapeutic agent to a subject, or application oradministration of a therapeutic agent to a cell or tissue from asubject, who has a diseases or disorder, has a symptom of a disease ordisorder, or is at risk of (or susceptible to) a disease or disorder,with the purpose of curing, inhibiting, healing, alleviating, relieving,altering, remedying, ameliorating, improving, or affecting the diseaseor disorder, the symptom of the disease or disorder, or the risk of (orsusceptibility to) the disease or disorder. As used herein, a“therapeutic agent” or “compound” includes, but is not limited to, smallmolecules, peptides, peptidomimetics, polypeptides, RNA interferingagents, e.g., siRNA molecules, antibodies, ribozymes, and antisenseoligonucleotides.

As described herein, cancer in subjects is associated with a change,e.g., an increase in the amount and/or activity, or a change in thestructure, of one or more markers listed in Table 2 (e.g., a marker thatwas shown to be increased in cancer), and/or a decrease in the amountand/or activity, or a change in the structure of one or more markerslisted in Table 1 (e.g., a marker that was shown to be decreased incancer). While, as discussed above, some of these changes in amount,structure, and/or activity, result from occurrence of the cancer, othersof these changes induce, maintain, and promote the cancerous state ofcancer, cells. Thus, cancer, characterized by an increase in the amountand/or activity, or a change in the structure, of one or more markerslisted in Table 2 (e.g., a marker that is shown to be increased incancer), can be inhibited by inhibiting amount, e.g., expression orprotein level, and/or activity of those markers. Likewise, cancercharacterized by a decrease in the amount and/or activity, or a changein the structure, of one or more markers listed in Table 1 (e.g., amarker that is shown to be decreased in cancer), can be inhibited byenhancing amount, e.g., expression or protein level, and/or activity ofthose markers

Accordingly, another aspect of the invention pertains to methods fortreating a subject suffering from cancer. These methods involveadministering to a subject a compound which modulates amount and/oractivity of one or more markers of the invention. For example, methodsof treatment or prevention of cancer include administering to a subjecta compound which decreases the amount and/or activity of one or moremarkers listed in Table 2 (e.g., a marker that was shown to be increasedin cancer). Compounds, e.g., antagonists, which may be used to inhibitamount and/or activity of a marker listed in Table 2, to thereby treator prevent cancer include antibodies (e.g., conjugated antibodies),small molecules, RNA interfering agents, e.g., siRNA molecules,ribozymes, and antisense oligonucleotides. In one embodiment, anantibody used for treatment is conjugated to a toxin, a chemotherapeuticagent, or radioactive particles.

Methods of treatment or prevention of cancer also include administeringto a subject a compound which increases the amount and/or activity ofone or more markers listed in Table 1 (e.g., a marker that was shown tobe decreased in cancer). Compounds, e.g., agonists, which may be used toincrease expression or activity of a marker listed in Table 1, tothereby treat or prevent cancer include small molecules, peptides,peptoids, peptidomimetics, and polypeptides.

Small molecules used in the methods of the invention include those whichinhibit a protein-protein interaction and thereby either increase ordecrease marker amount and/or activity. Furthermore, modulators, e.g.,small molecules, which cause re-expression of silenced genes, e.g.,tumor suppressors, are also included herein. For example, such moleculesinclude compounds which interfere with DNA binding or methyltransferaseactivity.

An aptamer may also be used to modulate, e.g., increase or inhibitexpression or activity of a marker of the invention to thereby treat,prevent or inhibit cancer. Aptamers are DNA or RNA molecules that havebeen selected from random pools based on their ability to bind othermolecules. Aptamers may be selected which bind nucleic acids orproteins.

VII. SCREENING ASSAYS

The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., proteins, peptides, peptidomimetics,peptoids, small molecules or other drugs) which (a) bind to a marker ofthe invention, or (b) have a modulatory (e.g., stimulatory orinhibitory) effect on the activity of a marker of the invention or, morespecifically, (c) have a modulatory effect on the interactions of amarker of the invention with one or more of its natural substrates(e.g., peptide, protein, hormone, co-factor, or nucleic acid), or (d)have a modulatory effect on the expression of a marker of the invention.Such assays typically comprise a reaction between the marker and one ormore assay components. The other components may be either the testcompound itself, or a combination of test compound and a natural bindingpartner of the marker. Compounds identified via assays such as thosedescribed herein may be useful, for example, for modulating, e.g.,inhibiting, ameliorating, treating, or preventing cancer.

The test compounds of the present invention may be obtained from anyavailable source, including systematic libraries of natural and/orsynthetic compounds. Test compounds may also be obtained by any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994,J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam, 1997, AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a marker of the invention orbiologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to a marker of the invention or biologically active portionthereof. Determining the ability of the test compound to directly bindto a marker can be accomplished, for example, by coupling the compoundwith a radioisotope or enzymatic label such that binding of the compoundto the marker can be determined by detecting the labeled marker compoundin a complex. For example, compounds (e.g., marker substrates) can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, assay components can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In another embodiment, the invention provides assays for screeningcandidate or test compounds which modulate the activity of a marker ofthe invention or a biologically active portion thereof. In alllikelihood, the marker can, in vivo, interact with one or moremolecules, such as, but not limited to, peptides, proteins, hormones,cofactors and nucleic acids. For the purposes of this discussion, suchcellular and extracellular molecules are referred to herein as “bindingpartners” or marker “substrate”.

One necessary embodiment of the invention in order to facilitate suchscreening is the use of the marker to identify its natural in vivobinding partners. There are many ways to accomplish this which are knownto one skilled in the art. One example is the use of the marker proteinas “bait protein” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al, 1993, Cell 72:223-232;Madura et al, 1993, J. Biol. Chem. 268:12046-12054; Bartel et al, 1993,Biotechniques 14:920-924; Iwabuchi et al, 1993 Oncogene 8:1693-1696;Brent WO94/10300) in order to identify other proteins which bind to orinteract with the marker (binding partners) and, therefore, are possiblyinvolved in the natural function of the marker. Such marker bindingpartners are also likely to be involved in the propagation of signals bythe marker or downstream elements of a marker-mediated signalingpathway. Alternatively, such marker binding partners may also be foundto be inhibitors of the marker.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that encodes a marker proteinfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a marker-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be readily detected and cell colonies containingthe functional transcription factor can be isolated and used to obtainthe cloned gene which encodes the protein which interacts with themarker protein.

In a further embodiment, assays may be devised through the use of theinvention for the purpose of identifying compounds which modulate (e.g.,affect either positively or negatively) interactions between a markerand its substrates and/or binding partners. Such compounds can include,but are not limited to, molecules such as antibodies, peptides,hormones, oligonucleotides, nucleic acids, and analogs thereof. Suchcompounds may also be obtained from any available source, includingsystematic libraries of natural and/or synthetic compounds. Thepreferred assay components for use in this embodiment is a cancer markeridentified herein, the known binding partner and/or substrate of same,and the test compound. Test compounds can be supplied from any source.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the marker and its bindingpartner involves preparing a reaction mixture containing the marker andits binding partner under conditions and for a time sufficient to allowthe two products to interact and bind, thus forming a complex. In orderto test an agent for inhibitory activity, the reaction mixture isprepared in the presence and absence of the test compound. The testcompound can be initially included in the reaction mixture, or can beadded at a time subsequent to the addition of the marker and its bindingpartner. Control reaction mixtures are incubated without the testcompound or with a placebo. The formation of any complexes between themarker and its binding partner is then detected. The formation of acomplex in the control reaction, but less or no such formation in thereaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the marker and its bindingpartner. Conversely, the formation of more complex in the presence ofcompound than in the control reaction indicates that the compound mayenhance interaction of the marker and its binding partner. The assay forcompounds that interfere with the interaction of the marker with itsbinding partner may be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either the marker or itsbinding partner onto a solid phase and detecting complexes anchored tothe solid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the markers andthe binding partners (e.g., by competition) can be identified byconducting the reaction in the presence of the test substance, i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with the marker and its interactive binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.,compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are briefly described below.

In a heterogeneous assay system, either the marker or its bindingpartner is anchored onto a solid surface or matrix, while the othercorresponding non-anchored component may be labeled, either directly orindirectly. In practice, microtitre plates are often utilized for thisapproach. The anchored species can be immobilized by a number ofmethods, either non-covalent or covalent, that are typically well knownto one who practices the art. Non-covalent attachment can often beaccomplished simply by coating the solid surface with a solution of themarker or its binding partner and drying. Alternatively, an immobilizedantibody specific for the assay component to be anchored can be used forthis purpose. Such surfaces can often be prepared in advance and stored.

In related embodiments, a fusion protein can be provided which adds adomain that allows one or both of the assay components to be anchored toa matrix. For example, glutathione-S-transferase/marker fusion proteinsor glutathione-S-transferase/binding partner can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedmarker or its binding partner, and the mixture incubated underconditions conducive to complex formation (e.g., physiologicalconditions). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound assay components, the immobilizedcomplex assessed either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of marker binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a markeror a marker binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated marker protein or target moleculescan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniquesknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). In certain embodiments, theprotein-immobilized surfaces can be prepared in advance and stored.

In order to conduct the assay, the corresponding partner of theimmobilized assay component is exposed to the coated surface with orwithout the test compound. After the reaction is complete, unreactedassay components are removed (e.g., by washing) and any complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.Where the non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the initially non-immobilizedspecies (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody). Depending upon theorder of addition of reaction components, test compounds which modulate(inhibit or enhance) complex formation or which disrupt preformedcomplexes can be detected.

In an alternate embodiment of the invention, a homogeneous assay may beused. This is typically a reaction, analogous to those mentioned above,which is conducted in a liquid phase in the presence or absence of thetest compound. The formed complexes are then separated from unreactedcomponents, and the amount of complex formed is determined. As mentionedfor heterogeneous assay systems, the order of addition of reactants tothe liquid phase can yield information about which test compoundsmodulate (inhibit or enhance) complex formation and which disruptpreformed complexes.

In such a homogeneous assay, the reaction products may be separated fromunreacted assay components by any of a number of standard techniques,including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, complexes of molecules may be separated from uncomplexedmolecules through a series of centrifugal steps, due to the differentsedimentation equilibria of complexes based on their different sizes anddensities (see, for example, Rivas, G., and Minton, A. P., TrendsBiochem Sci 1993 August; 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of thecomplex as compared to the uncomplexed molecules may be exploited todifferentially separate the complex from the remaining individualreactants, for example through the use of ion-exchange chromatographyresins. Such resins and chromatographic techniques are well known to oneskilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit. 11:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl.,699:499-525). Gel electrophoresis may also be employed to separatecomplexed molecules from unbound species (see, e.g., Ausubel et al(eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, NewYork. 1999). In this technique, protein or nucleic acid complexes areseparated based on size or charge, for example. In order to maintain thebinding interaction during the electrophoretic process, nondenaturinggels in the absence of reducing agent are typically preferred, butconditions appropriate to the particular interactants will be well knownto one skilled in the art. Immunoprecipitation is another commontechnique utilized for the isolation of a protein-protein complex fromsolution (see, e.g., Ausubel et al (eds.), In: Current Protocols inMolecular Biology, J. Wiley & Sons, New York. 1999). In this technique,all proteins binding to an antibody specific to one of the bindingmolecules are precipitated from solution by conjugating the antibody toa polymer bead that may be readily collected by centrifugation. Thebound assay components are released from the beads (through a specificproteolysis event or other technique well known in the art which willnot disturb the protein-protein interaction in the complex), and asecond immunoprecipitation step is performed, this time utilizingantibodies specific for the correspondingly different interacting assaycomponent. In this manner, only formed complexes should remain attachedto the beads. Variations in complex formation in both the presence andthe absence of a test compound can be compared, thus offeringinformation about the ability of the compound to modulate interactionsbetween the marker and its binding partner.

Also within the scope of the present invention are methods for directdetection of interactions between the marker and its natural bindingpartner and/or a test compound in a homogeneous or heterogeneous assaysystem without further sample manipulation. For example, the techniqueof fluorescence energy transfer may be utilized (see, e.g., Lakowicz etal, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No.4,868,103). Generally, this technique involves the addition of afluorophore label on a first ‘donor’ molecule (e.g., marker or testcompound) such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule (e.g., marker or testcompound), which in turn is able to fluoresce due to the absorbedenergy. Alternately, the ‘donor’ protein molecule may simply utilize thenatural fluorescent energy of tryptophan residues. Labels are chosenthat emit different wavelengths of light, such that the ‘acceptor’molecule label may be differentiated from that of the ‘donor’. Since theefficiency of energy transfer between the labels is related to thedistance separating the molecules, spatial relationships between themolecules can be assessed. In a situation in which binding occursbetween the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter). A test substancewhich either enhances or hinders participation of one of the species inthe preformed complex will result in the generation of a signal variantto that of background. In this way, test substances that modulateinteractions between a marker and its binding partner can be identifiedin controlled assays.

In another embodiment, modulators of marker expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of mRNA or protein, corresponding to a marker in the cell, isdetermined. The level of expression of mRNA or protein in the presenceof the candidate compound is compared to the level of expression of mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of marker expressionbased on this comparison. For example, when expression of marker mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of marker mRNA or protein expression.Conversely, when expression of marker mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of marker mRNA or protein expression. The level of marker mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting marker mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a marker protein can be furtherconfirmed in vivo, e.g., in a whole animal model for cancer, cellulartransformation and/or tumorigenesis. Animal models of lung cancer aredescribed in, for example, Tanaka, E., et al. (2003) Chest123:1248-1253., Jackson E L, et al (2001) Genes Dev 15(24):3243-8, andFisher G H, et al. (2001) Genes Dev 15(24):3249-62, the contents ofwhich are expressly incorporated herein by reference.

Additional animal based models of cancer are well known in the art(reviewed in Animal Models of Cancer Predisposition Syndromes, Hiai, Hand Hino, O (eds.) 1999, Progress in Experimental Tumor Research, Vol.35; Clarke A R Carcinogenesis (2000) 21:435-41) and include, forexample, carcinogen-induced tumors (Rithidech, K et al. Mutat Res (1999)428:33-39; Miller, M L et al. Environ Mol Mutagen (2000) 35:319-327),injection and/or transplantation of tumor cells into an animal, as wellas animals bearing mutations in growth regulatory genes, for example,oncogenes (e.g., ras) (Arbeit, J M et al. Am J Pathol (1993)142:1187-1197; Sinn, E et al. Cell (1987) 49:465-475; Thorgeirsson, S Set al Toxicol Lett (2000) 112-113:553-555) and tumor suppressor genes(e.g., p53) (Vooijs, M et al. Oncogene (1999) 18:5293-5303; Clark A RCancer Metast Rev (1995) 14:125-148; Kumar, T R et al. J Intern Med(1995) 238:233-238; Donehower, L A et al. (1992) Nature 356215-221).Furthermore, experimental model systems are available for the study of,for example, ovarian cancer (Hamilton, T C et al. Semin Oncol (1984)11:285-298; Rahman, N A et al. Mol Cell Endocrinol (1998) 145:167-174;Beamer, W G et al. Toxicol Pathol (1998) 26:704-710), gastric cancer(Thompson, J et al. Int J Cancer (2000) 86:863-869; Fodde, R et al.Cytogenet Cell Genet (1999) 86:105-111), breast cancer (Li, M et al.Oncogene (2000) 19:1010-1019; Green, J E et al. Oncogene (2000)19:1020-1027), melanoma (Satyamoorthy, K et al. Cancer Metast Rev(1999)18:401-405), and prostate cancer (Shirai, T et al. Mutat Res(2000) 462:219-226; Bostwick, D G et al. Prostate (2000) 43:286-294).Animal models described in, for example, Chin L. et al (1999) Nature400(6743):468-72, may also be used in the methods of the invention.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a marker modulating agent, a small molecule, anantisense marker nucleic acid molecule, a ribozyme, a marker-specificantibody, or fragment thereof, a marker protein, a marker nucleic acidmolecule, an RNA interfering agent, e.g., an siRNA molecule targeting amarker of the invention, or a marker-binding partner) can be used in ananimal model to determine the efficacy, toxicity, or side effects oftreatment with such an agent. Alternatively, an agent identified asdescribed herein can be used in an animal model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

VIII. PHARMACEUTICAL COMPOSITIONS

The small molecules, peptides, peptoids, peptidomimetics, polypeptides,RNA interfering agents, e.g., siRNA molecules, antibodies, ribozymes,and antisense oligonucleotides (also referred to herein as “activecompounds” or “compounds”) corresponding to a marker of the inventioncan be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the smallmolecules, peptides, peptoids, peptidomimetics, polypeptides, RNAinterfering agents, e.g., siRNA molecules, antibodies, ribozymes, orantisense oligonucleotides and a pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid corresponding to a marker of the invention. Such methods compriseformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention. Such compositions canfurther include additional active agents. Thus, the invention furtherincludes methods for preparing a pharmaceutical composition byformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention and one or more additionalactive compounds.

It is understood that appropriate doses of small molecule agents andprotein or polypeptide agents depends upon a number of factors withinthe knowledge of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of these agents will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the agent to have upon the nucleic acidmolecule or polypeptide of the invention. Small molecules include, butare not limited to, peptides, peptidomimetics, amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e., includingheteroorganic and organometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

Exemplary doses of a small molecule include milligram or microgramamounts per kilogram of subject or sample weight (e.g. about 1 microgramper kilogram to about 500 milligrams per kilogram, about 100 microgramsper kilogram to about 5 milligrams per kilogram, or about 1 microgramper kilogram to about 50 micrograms per kilogram).

As defined herein, a therapeutically effective amount of an RNAinterfering agent, e.g., siRNA, (i.e., an effective dosage) ranges fromabout 0.001 to 3,000 mg/kg body weight, preferably about 0.01 to 2500mg/kg body weight, more preferably about 0.1 to 2000, about 0.1 to 1000mg/kg body weight, 0.1 to 500 mg/kg body weight, 0.1 to 100 mg/kg bodyweight, 0.1 to 50 mg/kg body weight, 0.1 to 25 mg/kg body weight, andeven more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4to 7 mg/kg, or 5 to 6 mg/kg body weight. Treatment of a subject with atherapeutically effective amount of an RNA interfering agent can includea single treatment or, preferably, can include a series of treatments.In a preferred example, a subject is treated with an RNA interferingagent in the range of between about 0.1 to 20 mg/kg body weight, onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks.

Exemplary doses of a protein or polypeptide include gram, milligram ormicrogram amounts per kilogram of subject or sample weight (e.g. about 1microgram per kilogram to about 5 grams per kilogram, about 100micrograms per kilogram to about 500 milligrams per kilogram, or about 1milligram per kilogram to about 50 milligrams per kilogram). It isfurthermore understood that appropriate doses of one of these agentsdepend upon the potency of the agent with respect to the expression oractivity to be modulated. Such appropriate doses can be determined usingthe assays described herein. When one or more of these agents is to beadministered to an animal (e.g. a human) in order to modulate expressionor activity of a polypeptide or nucleic acid of the invention, aphysician, veterinarian, or researcher can, for example, prescribe arelatively low dose at first, subsequently increasing the dose until anappropriate response is obtained. In addition, it is understood that thespecific dose level for any particular animal subject will depend upon avariety of factors including the activity of the specific agentemployed, the age, body weight, general health, gender, and diet of thesubject, the time of administration, the route of administration, therate of excretion, any drug combination, and the degree of expression oractivity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediamine-tetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a polypeptide or antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium, and thenincorporating the required other ingredients from those enumeratedabove. In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches, and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes having monoclonal antibodies incorporated thereinor thereon) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into theepithelium). A method for lipidation of antibodies is described byCruikshank et al. (1997) J. Acquired Immune Deficiency Syndromes andHuman Retrovirology 14:193.

The nucleic acid molecules corresponding to a marker of the inventioncan be inserted into vectors and used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (U.S. Pat. No. 5,328,470),or by stereotactic injection (see, e.g., Chen et al., 1994, Proc. Natl.Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

The RNA interfering agents, e.g., siRNAs used in the methods of theinvention can be inserted into vectors. These constructs can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the vector can includethe RNA interfering agent, e.g., the siRNA vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

IX. PREDICTIVE MEDICINE

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningthe amount, structure, and/or activity of polypeptides or nucleic acidscorresponding to one or more markers of the invention, in order todetermine whether an individual is at risk of developing cancer. Suchassays can be used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset of the cancer.

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds administered either to inhibitcancer or to treat or prevent any other disorder {i.e. in order tounderstand any carcinogenic effects that such treatment may have}) onthe amount, structure, and/or activity of a marker of the invention inclinical trials. These and other agents are described in further detailin the following sections.

A. Diagnostic Assays

1. Methods for Detection of Copy Number

Methods of evaluating the copy number of a particular marker orchromosomal region (e.g., an MCR) are well known to those of skill inthe art. The presence or absence of chromosomal gain or loss can beevaluated simply by a determination of copy number of the regions ormarkers identified herein.

Methods for evaluating copy number of encoding nucleic acid in a sampleinclude, but are not limited to, hybridization-based assays. Forexample, one method for evaluating the copy number of encoding nucleicacid in a sample involves a Southern Blot. In a Southern Blot, thegenomic DNA (typically fragmented and separated on an electrophoreticgel) is hybridized to a probe specific for the target region. Comparisonof the intensity of the hybridization signal from the probe for thetarget region with control probe signal from analysis of normal genomicDNA (e.g., a non-amplified portion of the same or related cell, tissue,organ, etc.) provides an estimate of the relative copy number of thetarget nucleic acid. Alternatively, a Northern blot may be utilized forevaluating the copy number of encoding nucleic acid in a sample. In aNorthern blot, mRNA is hybridized to a probe specific for the targetregion. Comparison of the intensity of the hybridization signal from theprobe for the target region with control probe signal from analysis ofnormal mRNA (e.g., a non-amplified portion of the same or related cell,tissue, organ, etc.) provides an estimate of the relative copy number ofthe target nucleic acid.

An alternative means for determining the copy number is in situhybridization (e.g., Angerer (1987) Meth. Enzynmol 152: 649). Generally,in situ hybridization comprises the following steps: (1) fixation oftissue or biological structure to be analyzed; (2) prehybridizationtreatment of the biological structure to increase accessibility oftarget DNA, and to reduce nonspecific binding; (3) hybridization of themixture of nucleic acids to the nucleic acid in the biological structureor tissue; (4) post-hybridization washes to remove nucleic acidfragments not bound in the hybridization and (5) detection of thehybridized nucleic acid fragments. The reagent used in each of thesesteps and the conditions for use vary depending on the particularapplication.

Preferred hybridization-based assays include, but are not limited to,traditional “direct probe” methods such as Southern blots or in situhybridization (e.g., FISH and FISH plus SKY), and “comparative probe”methods such as comparative genomic hybridization (CGH), e.g.,cDNA-based or oligonucleotide-based CGH. The methods can be used in awide variety of formats including, but not limited to, substrate (e.g.membrane or glass) bound methods or array-based approaches.

In a typical in situ hybridization assay, cells are fixed to a solidsupport, typically a glass slide. If a nucleic acid is to be probed, thecells are typically denatured with heat or alkali. The cells are thencontacted with a hybridization solution at a moderate temperature topermit annealing of labeled probes specific to the nucleic acid sequenceencoding the protein. The targets (e.g., cells) are then typicallywashed at a predetermined stringency or at an increasing stringencyuntil an appropriate signal to noise ratio is obtained.

The probes are typically labeled, e.g., with radioisotopes orfluorescent reporters. Preferred probes are sufficiently long so as tospecifically hybridize with the target nucleic acid(s) under stringentconditions. The preferred size range is from about 200 bases to about1000 bases.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-I DNA is used to block non-specific hybridization.

In CGH methods, a first collection of nucleic acids (e.g., from asample, e.g., a possible tumor) is labeled with a first label, while asecond collection of nucleic acids (e.g., a control, e.g., from ahealthy cell/tissue) is labeled with a second label. The ratio ofhybridization of the nucleic acids is determined by the ratio of the two(first and second) labels binding to each fiber in the array. Wherethere are chromosomal deletions or multiplications, differences in theratio of the signals from the two labels will be detected and the ratiowill provide a measure of the copy number. Array-based CGH may also beperformed with single-color labeling (as opposed to labeling the controland the possible tumor sample with two different dyes and mixing themprior to hybridization, which will yield a ratio due to competitivehybridization of probes on the arrays). In single color CGH, the controlis labeled and hybridized to one array and absolute signals are read,and the possible tumor sample is labeled and hybridized to a secondarray (with identical content) and absolute signals are read. Copynumber difference is calculated based on absolute signals from the twoarrays. Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430,402; Methods in Molecular Biology, Vol. 33: In situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In oneembodiment, the hybridization protocol of Pinkel, et al. (1998) NatureGenetics 20: 207-211, or of Kallioniemi (1992) Proc. Natl. Acad Sci USA89:5321-5325 (1992) is used.

The methods of the invention are particularly well suited to array-basedhybridization formats. Array-based CGH is described in U.S. Pat. No.6,455,258, the contents of which are incorporated herein by reference.

In still another embodiment, amplification-based assays can be used tomeasure copy number. In such amplification-based assays, the nucleicacid sequences act as a template in an amplification reaction (e.g.,Polymerase Chain Reaction (PCR). In a quantitative amplification, theamount of amplification product will be proportional to the amount oftemplate in the original sample. Comparison to appropriate controls,e.g. healthy tissue, provides a measure of the copy number.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis, et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR may also be used in the methods of theinvention. In fluorogenic quantitative PCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan and sybr green.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Loss of heterozygosity (LOH) mapping (Wang, Z. C., et al. (2004) CancerRes 64(1):64-71; Seymour, A. B., et al. (1994) Cancer Res 54, 2761-4;Hahn, S. A., et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al.(1996) Genes Chromosomes Cancer 17, 88-93) may also be used to identifyregions of amplification or deletion.

2. Methods for Detection of Gene Expression

Marker expression level can also be assayed as a method for diagnosis ofcancer or risk for developing cancer. Expression of a marker of theinvention may be assessed by any of a wide variety of well known methodsfor detecting expression of a transcribed molecule or protein.Non-limiting examples of such methods include immunological methods fordetection of secreted, cell-surface, cytoplasmic, or nuclear proteins,protein purification methods, protein function or activity assays,nucleic acid hybridization methods, nucleic acid reverse transcriptionmethods, and nucleic acid amplification methods.

In preferred embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g. mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. Marker expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

Methods of detecting and/or quantifying the gene transcript (mRNA orcDNA made therefrom) using nucleic acid hybridization techniques areknown to those of skill in the art (see Sambrook et al. supra). Forexample, one method for evaluating the presence, absence, or quantity ofcDNA involves a Southern transfer as described above. Briefly, the mRNAis isolated (e.g. using an acid guanidinium-phenol-chloroform extractionmethod, Sambrook et al. supra.) and reverse transcribed to produce cDNA.The cDNA is then optionally digested and run on a gel in buffer andtransferred to membranes. Hybridization is then carried out using thenucleic acid probes specific for the target cDNA.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain a marker, and aprobe, under appropriate conditions and for a time sufficient to allowthe marker and probe to interact and bind, thus forming a complex thatcan be removed and/or detected in the reaction mixture. These assays canbe conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoringthe marker or probe onto a solid phase support, also referred to as asubstrate, and detecting target marker/probe complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, marker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which themarker or probe belongs. Well-known supports or carriers include, butare not limited to, glass, polystyrene, nylon, polypropylene,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above-mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

In a preferred embodiment, the probe, when it is the unanchored assaycomponent, can be labeled for the purpose of detection and readout ofthe assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect marker/probe complex formationwithout further manipulation or labeling of either component (marker orprobe), for example by utilizing the technique of fluorescence energytransfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169;Stavrianopoulos, et al, U.S. Pat. No. 4,868,103). A fluorophore label onthe first, ‘donor’ molecule is selected such that, upon excitation withincident light of appropriate wavelength, its emitted fluorescent energywill be absorbed by a fluorescent label on a second ‘acceptor’ molecule,which in turn is able to fluoresce due to the absorbed energy.Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, spatial relationships between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in the assayshould be maximal. An FET binding event can be conveniently measuredthrough standard fluorometric detection means well known in the art(e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S, andUrbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995,Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIAcore).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes may be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas, G., and Minton, A. P.,1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of themarker/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample, through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed SciAppl 1997 Oct. 10; 699(1-2):499-525). Gel electrophoresis may also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically preferred.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

In a particular embodiment, the level of mRNA corresponding to themarker can be determined both by in situ and by in vitro formats in abiological sample using methods known in the art. The term “biologicalsample” is intended to include tissues, cells, biological fluids andisolates thereof, isolated from a subject, as well as tissues, cells andfluids present within a subject. Many expression detection methods useisolated RNA. For in vitro methods, any RNA isolation technique thatdoes not select against the isolation of mRNA can be utilized for thepurification of RNA from cells (see, e.g., Ausubel et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York 1987-1999).Additionally, large numbers of tissue samples can readily be processedusing techniques well known to those of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155).

The isolated nucleic acid can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onepreferred diagnostic method for the detection of mRNA levels involvescontacting the isolated mRNA with a nucleic acid molecule (probe) thatcan hybridize to the mRNA encoded by the gene being detected. Thenucleic acid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding a marker ofthe present invention. Other suitable probes for use in the diagnosticassays of the invention are described herein. Hybridization of an mRNAwith the probe indicates that the marker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the markers of the present invention.

The probes can be full length or less than the full length of thenucleic acid sequence encoding the protein. Shorter probes areempirically tested for specificity. Preferably nucleic acid probes are20 bases or longer in length. (See, e.g., Sambrook et al. for methods ofselecting nucleic acid probe sequences for use in nucleic acidhybridization.) Visualization of the hybridized portions allows thequalitative determination of the presence or absence of cDNA.

An alternative method for determining the level of a transcriptcorresponding to a marker of the present invention in a sample involvesthe process of nucleic acid amplification, e.g., by rtPCR (theexperimental embodiment set forth in Mullis, 1987, U.S. Pat. No.4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci.USA, 88:189-193), self sustained sequence replication (Guatelli et al.,1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. Fluorogenic rtPCR may also be used in themethods of the invention. In fluorogenic rtPCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan and sybr green. Thesedetection schemes are especially useful for the detection of nucleicacid molecules if such molecules are present in very low numbers. Asused herein, amplification primers are defined as being a pair ofnucleic acid molecules that can anneal to 5′ or 3′ regions of a gene(plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10 to 30 nucleotides in length and flank a region from about 50 to200 nucleotides in length. Under appropriate conditions and withappropriate reagents, such primers permit the amplification of a nucleicacid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the cellsprior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absoluteexpression level of the marker, determinations may be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a subjectsample, to another sample, e.g., a non-cancerous sample, or betweensamples from different sources.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a marker,the level of expression of the marker is determined for 10 or moresamples of normal versus cancer cell isolates, preferably 50 or moresamples, prior to the determination of the expression level for thesample in question. The mean expression level of each of the genesassayed in the larger number of samples is determined and this is usedas a baseline expression level for the marker. The expression level ofthe marker determined for the test sample (absolute level of expression)is then divided by the mean expression value obtained for that marker.This provides a relative expression level.

Preferably, the samples used in the baseline determination will be fromcancer cells or normal cells of the same tissue type. The choice of thecell source is dependent on the use of the relative expression level.Using expression found in normal tissues as a mean expression score aidsin validating whether the marker assayed is specific to the tissue fromwhich the cell was derived (versus normal cells). In addition, as moredata is accumulated, the mean expression value can be revised, providingimproved relative expression values based on accumulated data.Expression data from normal cells provides a means for grading theseverity of the cancer state.

In another preferred embodiment, expression of a marker is assessed bypreparing genomic DNA or mRNA/cDNA (i.e. a transcribed polynucleotide)from cells in a subject sample, and by hybridizing the genomic DNA ormRNA/cDNA with a reference polynucleotide which is a complement of apolynucleotide comprising the marker, and fragments thereof cDNA can,optionally, be amplified using any of a variety of polymerase chainreaction methods prior to hybridization with the referencepolynucleotide. Expression of one or more markers can likewise bedetected using quantitative PCR (QPCR) to assess the level of expressionof the marker(s). Alternatively, any of the many known methods ofdetecting mutations or variants (e.g. single nucleotide polymorphisms,deletions, etc.) of a marker of the invention may be used to detectoccurrence of a mutated marker in a subject.

In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g. at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or morenucleotide residues) of a marker of the invention. If polynucleotidescomplementary to or homologous with are differentially detectable on thesubstrate (e.g. detectable using different chromophores or fluorophores,or fixed to different selected positions), then the levels of expressionof a plurality of markers can be assessed simultaneously using a singlesubstrate (e.g. a “gene chip” microarray of polynucleotides fixed atselected positions). When a method of assessing marker expression isused which involves hybridization of one nucleic acid with another, itis preferred that the hybridization be performed under stringenthybridization conditions.

In another embodiment, a combination of methods to assess the expressionof a marker is utilized.

Because the compositions, kits, and methods of the invention rely ondetection of a difference in expression levels or copy number of one ormore markers of the invention, it is preferable that the level ofexpression or copy number of the marker is significantly greater thanthe minimum detection limit of the method used to assess expression orcopy number in at least one of normal cells and cancerous cells.

3. Methods for Detection of Expressed Protein

The activity or level of a marker protein can also be detected and/orquantified by detecting or quantifying the expressed polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. These may include analyticbiochemical methods such as electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, and the like. A skilledartisan can readily adapt known protein/antibody detection methods foruse in determining whether cells express a marker of the presentinvention.

A preferred agent for detecting a polypeptide of the invention is anantibody capable of binding to a polypeptide corresponding to a markerof the invention, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

In a preferred embodiment, the antibody is labeled, e.g. aradio-labeled, chromophore-labeled, fluorophore-labeled, orenzyme-labeled antibody. In another embodiment, an antibody derivative(e.g. an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair {e.g. biotin-streptavidin}), or anantibody fragment (e.g. a single-chain antibody, an isolated antibodyhypervariable domain, etc.) which binds specifically with a proteincorresponding to the marker, such as the protein encoded by the openreading frame corresponding to the marker or such a protein which hasundergone all or a portion of its normal post-translationalmodification, is used.

Proteins from cells can be isolated using techniques that are well knownto those of skill in the art. The protein isolation methods employedcan, for example, be such as those described in Harlow and Lane (Harlowand Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

In one format, antibodies, or antibody fragments, can be used in methodssuch as Western blots or immunofluorescence techniques to detect theexpressed proteins. In such uses, it is generally preferable toimmobilize either the antibody or proteins on a solid support. Suitablesolid phase supports or carriers include any support capable of bindingan antigen or an antibody. Well-known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, protein isolated from cellscan be run on a polyacrylamide gel electrophoresis and immobilized ontoa solid phase support such as nitrocellulose. The support can then bewashed with suitable buffers followed by treatment with the detectablylabeled antibody. The solid phase support can then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on the solid support can then be detected by conventional means.Means of detecting proteins using electrophoretic techniques are wellknown to those of skill in the art (see generally, R. Scopes (1982)Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc., N.Y.).

In another preferred embodiment, Western blot (immunoblot) analysis isused to detect and quantify the presence of a polypeptide in the sample.This technique generally comprises separating sample proteins by gelelectrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind apolypeptide. The anti-polypeptide antibodies specifically bind to thepolypeptide on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-human antibodies) that specificallybind to the anti-polypeptide.

In a more preferred embodiment, the polypeptide is detected using animmunoassay. As used herein, an immunoassay is an assay that utilizes anantibody to specifically bind to the analyte. The immunoassay is thuscharacterized by detection of specific binding of a polypeptide to ananti-antibody as opposed to the use of other physical or chemicalproperties to isolate, target, and quantify the analyte.

The polypeptide is detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

Immunological binding assays (or immunoassays) typically utilize a“capture agent” to specifically bind to and often immobilize the analyte(polypeptide or subsequence). The capture agent is a moiety thatspecifically binds to the analyte. In a preferred embodiment, thecapture agent is an antibody that specifically binds a polypeptide. Theantibody (anti-peptide) may be produced by any of a number of means wellknown to those of skill in the art.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledpolypeptide or a labeled anti-antibody. Alternatively, the labelingagent may be a third moiety, such as another antibody, that specificallybinds to the antibody/polypeptide complex.

In one preferred embodiment, the labeling agent is a second humanantibody bearing a label. Alternatively, the second antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, e.g. asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom (1985) J. Immunol., 135: 2589-2542).

As indicated above, immunoassays for the detection and/or quantificationof a polypeptide can take a wide variety of formats well known to thoseof skill in the art.

Preferred immunoassays for detecting a polypeptide are eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of captured analyte is directly measured. In onepreferred “sandwich” assay, for example, the capture agent (anti-peptideantibodies) can be bound directly to a solid substrate where they areimmobilized. These immobilized antibodies then capture polypeptidepresent in the test sample. The polypeptide thus immobilized is thenbound by a labeling agent, such as a second human antibody bearing alabel.

In competitive assays, the amount of analyte (polypeptide) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte (polypeptide) displaced (or competed away) from acapture agent (anti-peptide antibody) by the analyte present in thesample. In one competitive assay, a known amount of, in this case, apolypeptide is added to the sample and the sample is then contacted witha capture agent. The amount of polypeptide bound to the antibody isinversely proportional to the concentration of polypeptide present inthe sample.

In one particularly preferred embodiment, the antibody is immobilized ona solid substrate. The amount of polypeptide bound to the antibody maybe determined either by measuring the amount of polypeptide present in apolypeptide/antibody complex, or alternatively by measuring the amountof remaining uncomplexed polypeptide. The amount of polypeptide may bedetected by providing a labeled polypeptide.

The assays of this invention are scored (as positive or negative orquantity of polypeptide) according to standard methods well known tothose of skill in the art. The particular method of scoring will dependon the assay format and choice of label. For example, a Western Blotassay can be scored by visualizing the colored product produced by theenzymatic label. A clearly visible colored band or spot at the correctmolecular weight is scored as a positive result, while the absence of aclearly visible spot or band is scored as a negative. The intensity ofthe band or spot can provide a quantitative measure of polypeptide.

Antibodies for use in the various immunoassays described herein, can beproduced as described herein.

In another embodiment, level (activity) is assayed by measuring theenzymatic activity of the gene product. Methods of assaying the activityof an enzyme are well known to those of skill in the art.

In vivo techniques for detection of a marker protein include introducinginto a subject a labeled antibody directed against the protein. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

Certain markers identified by the methods of the invention may besecreted proteins. It is a simple matter for the skilled artisan todetermine whether any particular marker protein is a secreted protein.In order to make this determination, the marker protein is expressed in,for example, a mammalian cell, preferably a human cell line,extracellular fluid is collected, and the presence or absence of theprotein in the extracellular fluid is assessed (e.g. using a labeledantibody which binds specifically with the protein).

The following is an example of a method which can be used to detectsecretion of a protein. About 8×10⁵ 293T cells are incubated at 37° C.in wells containing growth medium (Dulbecco's modified Eagle's medium{DMEM} supplemented with 10% fetal bovine serum) under a 5% (v/v) CO2,95% air atmosphere to about 60-70% confluence. The cells are thentransfected using a standard transfection mixture comprising 2micrograms of DNA comprising an expression vector encoding the proteinand 10 microliters of LipofectAMINE™ (GIBCO/BRL Catalog no. 18342-012)per well. The transfection mixture is maintained for about 5 hours, andthen replaced with fresh growth medium and maintained in an airatmosphere. Each well is gently rinsed twice with DMEM which does notcontain methionine or cysteine (DMEM-MC; ICN Catalog no. 16-424-54).About 1 milliliter of DMEM-MC and about 50 microcuries of Trans-³⁵S™reagent (ICN Catalog no. 51006) are added to each well. The wells aremaintained under the 5% CO₂ atmosphere described above and incubated at37° C. for a selected period. Following incubation, 150 microliters ofconditioned medium is removed and centrifuged to remove floating cellsand debris. The presence of the protein in the supernatant is anindication that the protein is secreted.

It will be appreciated that subject samples, e.g., a sample containingtissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinalfluid, urine, stool, bronchoalveolar lavage, and lung tissue, maycontain cells therein, particularly when the cells are cancerous, and,more particularly, when the cancer is metastasizing, and thus may beused in the methods of the present invention. The cell sample can, ofcourse, be subjected to a variety of well-known post-collectionpreparative and storage techniques (e.g., nucleic acid and/or proteinextraction, fixation, storage, freezing, ultrafiltration, concentration,evaporation, centrifugation, etc.) prior to assessing the level ofexpression of the marker in the sample. Thus, the compositions, kits,and methods of the invention can be used to detect expression of markerscorresponding to proteins having at least one portion which is displayedon the surface of cells which express it. It is a simple matter for theskilled artisan to determine whether the protein corresponding to anyparticular marker comprises a cell-surface protein. For example,immunological methods may be used to detect such proteins on wholecells, or well known computer-based sequence analysis methods (e.g. theSIGNALP program; Nielsen et al., 1997, Protein Engineering 10:1-6) maybe used to predict the presence of at least one extracellular domain(i.e. including both secreted proteins and proteins having at least onecell-surface domain). Expression of a marker corresponding to a proteinhaving at least one portion which is displayed on the surface of a cellwhich expresses it may be detected without necessarily lysing the cell(e.g. using a labeled antibody which binds specifically with acell-surface domain of the protein).

The invention also encompasses kits for detecting the presence of apolypeptide or nucleic acid corresponding to a marker of the inventionin a biological sample, e.g., a sample containing tissue, whole blood,serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool,bronchoalveolar lavage, and lung tissue. Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping cancer. For example, the kit can comprise a labeled compoundor agent capable of detecting a polypeptide or an mRNA encoding apolypeptide corresponding to a marker of the invention in a biologicalsample and means for determining the amount of the polypeptide or mRNAin the sample (e.g., an antibody which binds the polypeptide or anoligonucleotide probe which binds to DNA or mRNA encoding thepolypeptide). Kits can also include instructions for interpreting theresults obtained using the kit.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide corresponding to a marker of the invention; and, optionally,(2) a second, different antibody which binds to either the polypeptideor the first antibody and is conjugated to a detectable label.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit can furthercomprise components necessary for detecting the detectable label (e.g.,an enzyme or a substrate). The kit can also contain a control sample ora series of control samples which can be assayed and compared to thetest sample. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

4. Method for Detecting Structural Alterations

The invention also provides a method for assessing whether a subject isafflicted with cancer or is at risk for developing cancer by comparingthe structural alterations, e.g., mutations or allelic variants, of amarker in a cancer sample with the structural alterations, e.g.,mutations of a marker in a normal, e.g., control sample. The presence ofa structural alteration, e.g., mutation or allelic variant in the markerin the cancer sample is an indication that the subject is afflicted withcancer.

A preferred detection method is allele specific hybridization usingprobes overlapping the polymorphic site and having about 5, 10, 20, 25,or 30 nucleotides around the polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to allelic variants are attached to a solid phase support,e.g., a “chip”. Oligonucleotides can be bound to a solid support by avariety of processes, including lithography. For example a chip can holdup to 250,000 oligonucleotides (GeneChip, Affymetrix™). Mutationdetection analysis using these chips comprising oligonucleotides, alsotermed “DNA probe arrays” is described e.g., in Cronin et al. (1996)Human Mutation 7:244. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment. For example, theidentity of the allelic variant of the nucleotide polymorphism in the 5′upstream regulatory element can be determined in a single hybridizationexperiment.

In other detection methods, it is necessary to first amplify at least aportion of a marker prior to identifying the allelic variant.Amplification can be performed, e.g., by PCR and/or LCR (see Wu andWallace (1989) Genomics 4:560), according to methods known in the art.In one embodiment, genomic DNA of a cell is exposed to two PCR primersand amplification for a number of cycles sufficient to produce therequired amount of amplified DNA. In preferred embodiments, the primersare located between 150 and 350 base pairs apart.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., (1988) Bio/Technology 6:1197), andself-sustained sequence replication (Guatelli et al., (1989) Proc. Nat.Acad. Sci. 87:1874), and nucleic acid based sequence amplification(NABSA), or any other nucleic acid amplification method, followed by thedetection of the amplified molecules using techniques well known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers.

In one embodiment, any of a variety of sequencing reactions known in theart can be used to directly sequence at least a portion of a marker anddetect allelic variants, e.g., mutations, by comparing the sequence ofthe sample sequence with the corresponding reference (control) sequence.Exemplary sequencing reactions include those based on techniquesdeveloped by Maxam and Gilbert (Proc. Natl. Acad Sci USA (1977) 74:560)or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci. 74:5463). It isalso contemplated that any of a variety of automated sequencingprocedures may be utilized when performing the subject assays(Biotechniques (1995) 19:448), including sequencing by mass spectrometry(see, for example, U.S. Pat. No. 5,547,835 and international patentapplication Publication Number WO 94/16101, entitled DNA Sequencing byMass Spectrometry by H. Köster; U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/21822 entitledDNA Sequencing by Mass Spectrometry Via Exonuclease Degradation by H.Köster), and U.S. Pat. No. 5,605,798 and International PatentApplication No. PCT/US96/03651 entitled DNA Diagnostics Based on MassSpectrometry by H. Köster; Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159). It will be evident to one skilled in the art that, forcertain embodiments, the occurrence of only one, two or three of thenucleic acid bases need be determined in the sequencing reaction. Forinstance, A-track or the like, e.g., where only one nucleotide isdetected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Pat. No.5,580,732 entitled “Method of DNA sequencing employing a mixedDNA-polymer chain probe” and U.S. Pat. No. 5,571,676 entitled “Methodfor mismatch-directed in vitro DNA sequencing.”

In some cases, the presence of a specific allele of a marker in DNA froma subject can be shown by restriction enzyme analysis. For example, aspecific nucleotide polymorphism can result in a nucleotide sequencecomprising a restriction site which is absent from the nucleotidesequence of another allelic variant.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, thetechnique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing a control nucleic acid, which is optionallylabeled, e.g., RNA or DNA, comprising a nucleotide sequence of a markerallelic variant with a sample nucleic acid, e.g., RNA or DNA, obtainedfrom a tissue sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such asduplexes formed based on basepair mismatches between the control andsample strands. For instance, RNA/DNA duplexes can be treated with RNaseand DNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine whether the control andsample nucleic acids have an identical nucleotide sequence or in whichnucleotides they are different. See, for example, Cotton et al (1988)Proc. Natl. Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control or sample nucleicacid is labeled for detection.

In another embodiment, an allelic variant can be identified bydenaturing high-performance liquid chromatography (DHPLC) (Oefner andUnderhill, (1995) Am. J. Human Gen. 57:Suppl. A266). DHPLC usesreverse-phase ion-pairing chromatography to detect the heteroduplexesthat are generated during amplification of PCR fragments fromindividuals who are heterozygous at a particular nucleotide locus withinthat fragment (Oefner and Underhill (1995) Am. J. Human Gen. 57:Suppl.A266). In general, PCR products are produced using PCR primers flankingthe DNA of interest. DHPLC analysis is carried out and the resultingchromatograms are analyzed to identify base pair alterations ordeletions based on specific chromatographic profiles (see O'Donovan etal. (1998) Genomics 52:44-49).

In other embodiments, alterations in electrophoretic mobility are usedto identify the type of marker allelic variant. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci. USA 86:2766,see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) GenetAnal Tech Appl 9:73-79). Single-stranded DNA fragments of sample andcontrol nucleic acids are denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence and the resulting alteration in electrophoretic mobilityenables the detection of even a single base change. The DNA fragmentsmay be labeled or detected with labeled probes. The sensitivity of theassay may be enhanced by using RNA (rather than DNA), in which thesecondary structure is more sensitive to a change in sequence. Inanother preferred embodiment, the subject method utilizes heteroduplexanalysis to separate double stranded heteroduplex molecules on the basisof changes in electrophoretic mobility (Keen et al. (1991) Trends Genet.7:5).

In yet another embodiment, the identity of an allelic variant of apolymorphic region is obtained by analyzing the movement of a nucleicacid comprising the polymorphic region in polyacrylamide gels containinga gradient of denaturant is assayed using denaturing gradient gelelectrophoresis (DGGE) (Myers et al (1985) Nature 313:495). When DGGE isused as the method of analysis, DNA will be modified to insure that itdoes not completely denature, for example by adding a GC clamp ofapproximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

Examples of techniques for detecting differences of at least onenucleotide between two nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrally(allele-specific probes) and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl.Acad. Sci. USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res.6:3543). Such allele specific oligonucleotide hybridization techniquesmay be used for the simultaneous detection of several nucleotide changesin different polymorphic regions of marker. For example,oligonucleotides having nucleotide sequences of specific allelicvariants are attached to a hybridizing membrane and this membrane isthen hybridized with labeled sample nucleic acid. Analysis of thehybridization signal will then reveal the identity of the nucleotides ofthe sample nucleic acid.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the allelic variant of interest in the center of the molecule(so that amplification depends on differential hybridization) (Gibbs etal (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end ofone primer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238; Newton etal. (1989) Nucl. Acids Res. 17:2503). This technique is also termed“PROBE” for Probe Oligo Base Extension. In addition it may be desirableto introduce a novel restriction site in the region of the mutation tocreate cleavage-based detection (Gasparini et al (1992) Mol. Cell.Probes 6:1).

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., (1988) Science241:1077-1080. The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., (1990)Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927. In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

The invention further provides methods for detecting single nucleotidepolymorphisms in a marker. Because single nucleotide polymorphismsconstitute sites of variation flanked by regions of invariant sequence,their analysis requires no more than the determination of the identityof the single nucleotide present at the site of variation and it isunnecessary to determine a complete gene sequence for each subject.Several methods have been developed to facilitate the analysis of suchsingle nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site(Cohen, D. et al French Patent 2,650,840; PCT Appln. No. WO91/02087). Asin the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assayingpolymorphic sites in DNA have been described (Komher, J. S. et al.,(1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, B. P., (1990) Nucl.Acids Res. 18:3671; Syvanen, A.-C., et al., (1990) Genomics 8:684-692;Kuppuswamy, M. N. et al., (1991) Proc. Natl. Acad. Sci. (U.S.A.)88:1143-1147; Prezant, T. R. et al., (1992) Hum. Mutat. 1:159-164;Ugozzoli, L. et al., (1992) GATA 9:107-112; Nyren, P. (1993) et al.,Anal. Biochem. 208:171-175). These methods differ from GBA™ in that theyall rely on the incorporation of labeled deoxynucleotides todiscriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidecan result in signals that are proportional to the length of the run(Syvanen, A. C., et al., (1993) Amer. J. Hum. Genet. 52:46-59).

For determining the identity of the allelic variant of a polymorphicregion located in the coding region of a marker, yet other methods thanthose described above can be used. For example, identification of anallelic variant which encodes a mutated marker can be performed by usingan antibody specifically recognizing the mutant protein in, e.g.,immunohistochemistry or immunoprecipitation. Antibodies to wild-typemarker or mutated forms of markers can be prepared according to methodsknown in the art.

Alternatively, one can also measure an activity of a marker, such asbinding to a marker ligand. Binding assays are known in the art andinvolve, e.g., obtaining cells from a subject, and performing bindingexperiments with a labeled ligand, to determine whether binding to themutated form of the protein differs from binding to the wild-type of theprotein.

B. Pharmacogenomics

Agents or modulators which have a stimulatory or inhibitory effect onamount and/or activity of a marker of the invention can be administeredto individuals to treat (prophylactically or therapeutically) cancer inthe subject. In conjunction with such treatment, the pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) of theindividual may be considered. Differences in metabolism of therapeuticscan lead to severe toxicity or therapeutic failure by altering therelation between dose and blood concentration of the pharmacologicallyactive drug. Thus, the pharmacogenomics of the individual permits theselection of effective agents (e.g., drugs) for prophylactic ortherapeutic treatments based on a consideration of the individual'sgenotype. Such pharmacogenomics can further be used to determineappropriate dosages and therapeutic regimens. Accordingly, the amount,structure, and/or activity of the invention in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant variations in theresponse to drugs due to altered drug disposition and abnormal action inaffected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266. Ingeneral, two types of pharmacogenetic conditions can be differentiated.Genetic conditions transmitted as a single factor altering the way drugsact on the body are referred to as “altered drug action.” Geneticconditions transmitted as single factors altering the way the body actson drugs are referred to as “altered drug metabolism”. Thesepharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD)deficiency is a common inherited enzymopathy in which the main clinicalcomplication is hemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some subjectsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the amount, structure, and/or activity of a marker of theinvention in an individual can be determined to thereby selectappropriate agent(s) for therapeutic or prophylactic treatment of theindividual. In addition, pharmacogenetic studies can be used to applygenotyping of polymorphic alleles encoding drug-metabolizing enzymes tothe identification of an individual's drug responsiveness phenotype.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a modulator ofamount, structure, and/or activity of a marker of the invention.

C. Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on amount,structure, and/or activity of a marker of the invention can be appliednot only in basic drug screening, but also in clinical trials. Forexample, the effectiveness of an agent to affect marker amount,structure, and/or activity can be monitored in clinical trials ofsubjects receiving treatment for cancer. In a preferred embodiment, thepresent invention provides a method for monitoring the effectiveness oftreatment of a subject with an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, antibody, nucleic acid, antisensenucleic acid, ribozyme, small molecule, RNA interfering agent, or otherdrug candidate) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the amount, structure, and/or activity of one ormore selected markers of the invention in the pre-administration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the amount, structure, and/or activity of themarker(s) in the post-administration samples; (v) comparing the amount,structure, and/or activity of the marker(s) in the pre-administrationsample with the amount, structure, and/or activity of the marker(s) inthe post-administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent can be desirable to increaseamount and/or activity of the marker(s) to higher levels than detected,i.e., to increase the effectiveness of the agent. Alternatively,decreased administration of the agent can be desirable to decreaseamount and/or activity of the marker(s) to lower levels than detected,i.e., to decrease the effectiveness of the agent.

EXAMPLES

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,figures, Sequence Listing, patents and published patent applicationscited throughout this application are hereby incorporated by reference.

Example 1 A. Materials and Methods

Cell Lines and Primary Tumors. All the primary tumors were acquired fromthe Cooperative Human Tissue Network (CHTN, Philadelphia, Pa.) andBrigham and Women Hospital Tissue Bank (Boston, Mass.). All the celllines were obtained from the American Type Culture Collection (ATCC,Manassas, Va.). The histology was confirmed. The characteristics of thecell lines and of the primary tumors are reported in Tables 4 and 5,respectively.

Array-CGH profiling on cDNA microarrays. Genomic DNAs from cell linesand primary tumors were extracted according to the manufacturerinstructions (Gentra System Lie, Minneapolis, Minn.). Genomic DNA wasfragmented and random-prime labeled according to published protocols(Pollack, J. R., et al. (1999a) Nat Genet 23, 41-46.) withmodifications. For details, see Aguirre, A. J., et al. (2004) Proc NatlAcad Sci USA 101, 9067-72. Labeled DNAs were hybridized to either humancDNA microarrays or oligo microarrays. The cDNA microarrays contain14,160 cDNA clones (Agilent Technologies, Human 1 clone set) with 13,281mappable clones, for which approximately 11,211 unique map positionswere defined (NCBI, Build 35). The median interval between mappedelements is 72.7 kilobase, 94.1% of intervals are less than 1 Mb, and98.9% are less than 3 Mb. The oligo array contains 22K oligonucleotides(Agilent Technologies). All probes were subjected to BLAST alignmentwith the latest draft of human/mouse genome sequence (NCBI, Build 35).Based on the BLAST results, un-mappable probes were eliminated, whichwere arbitrarily defined as alignment length of best hit less than 55bp; and un-informative probes, which were identified by having a secondbest hit score of 95% or higher on the alignment length of the best hit.These criteria selected 16022 informative probes for the human array.The median interval between mapped elements is 54.7 kilobase, 96.7% ofintervals are less than 1 Mb, and 99.5% are less than 3 Mb.

Fluorescence ratios of scanned images of the arrays were calculated andthe raw array-CGH profiles were processed to identify statisticallysignificant transitions in copy number using a segmentation algorithmwhich employs permutation to determine the significance of change pointsin the raw data (Olshen, A. B., et al. (2004) Biostatistics5(4):557-72). Each segment is assigned a Log₂ ratio that is the medianof the contained probes. The data is centered by the tallest mode in thedistribution of the segmented values. After mode-centering, gains andlosses were defined as Log₂ ratios of greater than or equal to +0.11 or−0.11 (+/−4 standard deviations of the middle 50% quantile of data), andamplification and deletion as a ratio greater than 0.4 or less than−0.4, respectively. For the cDNA dataset, gains and losses were definedas Log₂ ratios of greater than or equal to +0.13 or −0.13 (+/−4 standarddeviations of the middle 50% quantile of data), and amplification anddeletion as a ratio greater than 0.55 or less than −0.5, respectively.The permutation test to compare the squamous cell carcinoma and theadenocarcinoma oncogenomes was performed as follows. The segmented arrayCGH data were divided into squamous cell carcinoma and theadenocarcinoma groups. Thresholds 0.1 (over) and −0.1 (under) were usedfor obtaining genome changes. At each probe, all samples exceeding thethresholds were counted for the squamous cell carcinoma and theadenocarcinoma groups, respectively. The scores were then calculated asfollowing:score.over=(squamous.over−adeno.over)²/(squamous.over+adeno.over)score.under=(squamous.under−adeno.under)²/(squamous.under+adeno.under)

The permutations were performed 1000 times by scrambling sample grouplabels. The scores were calculated as above and the peak score for allprobes was maintained for each permutation. The statisticallysignificant probes were identified by comparing the score among 1000peak scores from permutation testing with a 5% false positive rate asthe cut off.

The comparison between amplificatioii/deletion in squamous andadenocarcinomas was performed as follows. A custom-made algorithm wasdesigned to identify all the probes, on the segmented data, that wereabove a Log₂ ratio of 0.6 in at least 15% of the samples in one tumorsubtype and absent in the other dataset. For the deletions, thealgorithm searched for all the probes on the segmented data that werebelow a Log₂ ratio of −0.6 in at least 15% of the samples in one tumorsubtype and absent in the other dataset.

Automated locus definition. Minimal Common Regions (MCRs) were definedby an automated algorithm applied to the segmented data based on thefollowing rules:

1. Segments above 0.4 or below −0.4 (0.5 and 0.55 for cDNA) areidentified as altered.

2. If two or more altered segments are adjacent in a single profile orseparated by less than 500 KB, the entire region spanned by the segmentsis considered to be an altered span.

3. Highly altered segments or spans that are shorter than 20 MB areretained as “informative spans” for defining discrete locus boundaries.Longer regions are not discarded, but are not included in defining locusboundaries.

4. Informative spans are compared across samples to identify overlappinggroups of positive-value or negative-value segments; each group iscalled an “overlap group”.

5. Overlap groups are divided into separate groups wherever therecurrence rate falls below 25% of the peak recurrence for the wholegroup. Overlap groups are divided into separate groups wherever therecurrence rate falls below 25% of the peak recurrence for the wholegroup. Recurrence is calculated by counting the number of samples withalteration at high threshold (0.4, −0.4).

6. Minimal common regions (MCRs) are defined as contiguous spans havingat least 75% of the peak recurrence. If two MCRs are separated by a gapof only one probe position, they are joined. If there are more than 3MCRs in a locus, the whole region is reported as a single complex MCR.

MCR Characterization. For each MCR, the peak segment value isidentified. Recurrence of gain or loss is calculated across all samplesbased on the lower thresholds previously defined (˜+/−0.11). As anadditional measure of recurrence independent of thresholds for segmentvalue or length, Median Aberration (MA) is calculated for each probeposition by taking the median of all segment values above zero foramplified regions, below zero for deleted regions. This pair of valuesis compared to the distribution of values obtained after permuting theprobe labels independently in each sample profile. Where the magnitudeof the MA exceeds 95% of the permuted averages, the region is marked assignificantly gained or lost, and this is used in the voting system forprioritization. The number of known genes is counted based on the July2003 human assembly at NCBI (build 35).

Complexity analysis. The change-point statistical model used for copynumber inference data (Olshen, A. B., et al. (2004) Biostatistics5(4):557-72) defines a contiguous genomic region of uniform copy numberas a segment: each segment contains a copy number different from itsneighboring segments and so transitions between adjacent segmentsdelineate transition in copy number. The number of segments and the sizeof these segments are indicators of frequency of copy number alterationsacross the genome. Only segments above or below 0.4 were considered inthis analysis.

Sequencing. Primers for PCR amplification and sequencing of the FGFR1tyrosine kinase domain (Celera accession hCT1964964, exons 12-18) weredesigned as previously described (Bardelli, A., et al. (2003) Science300, 94.). Forward and reverse sequences of PCR-amplified exons wereacquired from the six primary lung tumors and the two lung cancer celllines harboring the 8p11 genomic amplification. Chromatograms wereassembled using Sequencher (Gene Codes Corporation, Ann Arbor, Mich.)and manually compared to the NCBI reference sequence for FGFR1 foridentification of possible mutations (see Table 6).

Quantitative PCR (QPCR) verification. PCR primers were designed toamplify products of 100-150 bp within target and control sequences (seeTable 6). Primers for control sequences in each cell line were designedwithin a region of euploid copy number as shown by array-CGH analysis.Quantitative PCR was performed, by monitoring in real-time, the increasein fluorescence of SYBR Green dye (Qiagen, Valencia, Calif.) with an ABI7700 sequence detection system (Perkin Elmer Life Sciences, Boston,Mass.). Relative gene copy number was calculated by the standard curvemethod (Ginzinger, D. G. (2002) Exp Hematol 30, 503-512). Estimates ofgene dosage were made relative to the most common copy number within asample. For PCR validation, abundant Line elements were used as areference against which to compare copy number alterations. Based onprevious experience with other datasets (Aguirre, A. J., et al. (2004)Proc Natl Acad Sci USA 101, 9067-9072; Brennan, C., et al. (2004) CancerRes 64, 4744-4748), a threshold of 2 (as relative gene dosage) was usedas a cutoff of alteration. For validation of expression, RNA-specificPCR primers were designed to amplify products of 100-150 bp across exons(see Table 6). As reference, normal lung RNA was used (Ambion, Austin,Tex.).

Expression Profiling on Affymetrix GeneChip. Biotinylated target cRNAwas generated from total sample RNA and hybridized to humanoligonucleotide probe arrays (U133Plus 2.0, Affymetrix, Santa Clara,Calif.) according to standard protocols (Golub, T. R., et al. (1999)Science 286, 531-537). Expression values were normalized in dChip (Li,C., and Wong, W. H. (2001) Proc Natl Acad Sci USA 98, 31-36) and thenfor each gene were standardized by Log₂ ratio to the median of thecohort. Twenty-eight cell lines, twenty-nine tumors, as well as normaltissue adjacent to tumor tissue were analyzed on these oligo arrays.

Integrated copy number and expression analysis. Array-CGH data wasinterpolated such that each expression value can be mapped to itscorresponding copy number value. For each gene position, the sampleswere grouped based on whether array-CGH showed altered copy number ornot based on interpolated CGH value. The effect of gene dosage onexpression was measured by calculating a gene weight defined as thedifference of the means of the expression value in the altered andunaltered sample groups divided by the sum of the standard deviations ofthe expression values in altered and unaltered sample groups (Aguirre,A. J., et al. (2004) Proc Natl Acad Sci USA 101, 9067-9072; Hyman, E.,et al (2002) Cancer Res 62, 6240-6245). The significance of the weightfor each gene was estimated by permuting the sample labels 10,000 timesand applying an alpha threshold of 0.05.

Fluorescence in situ hybridization (FISH). Metaphase spread slides wereprepared following standard protocols (Protopopov, A. I., et al. (1996)Chromosome Res 4, 443-447). Frozen tissue sections (4 μm) werepre-treated according to manufacturer's protocol (Frozen Tissue Prep forFISH, Vysis, Downers Grove, Ill.). The BACs RP11-138J2 (at 27 MB, CHR8), RP11-265K5 (from HTPAP to LETM2 on CHR 8; BAC #1), RP11-100B16(covering FGFR1 and FLJ43582 on CHR 8; BAC#2) and RP11-90P5 (coveringfrom LSM1 to DDHD2 on CHR 8; BAC#3) were used for the hybridizations.The probes for the FISH analysis were labeled using nick translation,according to the manufacturer's instructions (Roche MolecularBiochemicals, Indianapolis, Ind.) with either biotin-14-dATP ordigoxigenin-11-dUTP. Biotinylated probes were detected usingCy3-conjugated avidin (Accurate Chemical, Westbury, N.Y.). Fordigoxigenin-labeled probes, antidigoxigenin-FITC Fab fragments (EnzoLife Sciences, Farmingdale, N.Y.) was used. Slides were counterstainedwith 5 μg/ml 4′,6-diamidino-2-phenylindole (Merck, Wilmington, Del.) andmounted in Vectashield antifade medium (Vector Laboratories, Burlingame,Calif.). FISH signals acquisition and spectral analysis was performedusing filter sets and software developed by Applied Spectral Imaging(Carlsbad, Calif.).

B. Results

Lung cancer is the leading cause of cancer related mortality in theUnited States, accounting for more than one-fourth of all cancerfatalities in 2004. Distinctive clinical and pathobiological featuresserve to classify lung cancers into two major subtypes: small cell lungcancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC constitutes75% of lung cancer cases and is subdivided further into three majorhistologically distinct subtypes including adenocarcinoma, squamous cellcarcinoma and large cell carcinoma. Adenocarcinoma and squamouscarcinoma subtypes represent over 85% of NSCLC cases. While these NSCLCsubtypes exhibit distinct clinical and pathological characteristics, thetreatment approaches have remained generic and largely ineffectivedespite advances in cytotoxic drugs, radiotherapy, and clinicalmanagement. The cure rate for advanced NSCLC cancer remains among themost dismal in oncology. For all stages of NSCLC, the 5-year survivalrate has remained fixed at 15% for the last 15 years. The recent successof molecularly targeted therapies for a limited number of cases withspecific cancer genotypes (Lynch, T. J., et al. (2004). N Engl J Med350, 2129-2139; Paez, J. G., et al. (2004) Science 304, 1497-1500) hassolidified the view that a more detailed knowledge of the spectrum ofgenetic lesions in lung cancer will in turn lead to meaningfultherapeutic progress.

To date, the majority of lung cancer genetic studies have catalogedmutations or promoter methylation status of known cancer genes,performed genome-wide loss-of-heterozygosity surveys, and applied CGH toaudit regional copy number alterations (CNAs) on metaphase chromosomesor small-scale BAC arrays. These concerted efforts have identified acore set of lesions including K-RAS activation mutations andloss-of-function mutations in the p53 and Rb pathways (Minna, J. D., etal (2002) Cancer Cell 1, 49-52). At the same time, the observed highnumber of recurrent chromosomal aberrations, particularly amplificationsand deletions, belies that only a small fraction of the lung cancergenes has been identified to date.

Integrated CGH and expression profiling have emerged as a highlyeffective approach to cancer gene discovery, capable of providing agenome-wide view of the regional gains and losses throughout the cancergenome (Kallioniemi, O. P., et al. (1994) Genes Chromosomes Cancer 10,231-243) and associated copy number-driven changes in gene expression(Aguirre, A. J., et al. (2004) Proc Natl Acad Sci USA 101, 9067-9072;Pollack, J. R., et al. (1999b) Nat Genet. 23, 41-46; Pollack, J. R., etal (2002) Proc Natl Acad Sci USA 99, 12963-12968). In the case of NSCLC,chromosomal CGH studies have revealed recurrent gains at 1q31, 3q25-27,5p13-14 and 8q23-24 and deletions at 3p21, 8p22, 9p21-22, 13q22, and17p12-13 (Balsara, B. R., and Testa, J. R. (2002) Oncogene 21,6877-6883; Bjorkqvist, A. M., et al (1998) Br J Cancer 77, 260-269; Luk,C., et al. (2001) Cancer Genet Cytogenet 125, 87-99; Pei, J., et al.(2001) Genes Chromosomes Cancer 31, 282-287; Petersen, I., et al. (1997)Cancer Res 57, 2331-2335). A recent limited array CGH survey of knowngenes/loci possibly contributing to lung cancer has demonstrated theutility of this approach by confirming recurrent chromosome 3p deletionsand 3q gains, and identified PIK3CA as a resident of chromosome 3qamplicon (Massion, P. P., et al. (2002) Cancer Res 62, 3636-3640), agene shown subsequently to harbor activating point mutations in somelung cancer cases (Samuels, Y., et al. (2004) Science 304 554).

In the microarray format, the resolution of CGH is dictated by thenumber and quality of mapped probes positioned along the genome(Albertson, D. G., and Pinkel, D. (2003) Hum Mol Genet. 12, 145R-152).As described herein, high-density cDNA- and oligonucleotide-based arrayswere used to conduct high-resolution surveys of CNAs present in awell-defined collection of primary adenocarcinomas and squamous cellcarcinomas and in a panel of established NSCLC cell lines. Theapplication of novel custom bioinformatics tools, along with integrationof expression profiles and public databases, has yielded a dramaticincrease in the number of recurrent amplifications and deletionsdetectable in the NSCLC genome. The high degree of NSCLC genomiccomplexity, the recurrent nature of these lesions, and preliminaryfunctional characterization of resident genes points to a large numberof relevant oncogenic events, creating therapeutic and diagnosticcompositions of matter and methods of using the same.

Identification of Known and Novel CNAs in the NSCLC Genome.

Forty-five tumors, frozen at the time of the initial resection andverified to be adenocarcinomas (n=19) or squamous cell carcinomas (n=26)and possessing greater than 70% tumor cellularity were subjected togenome-wide CGH profiling (Table 4). In addition to the primary tumors,CGH and expression profiling was performed on a panel of 34 NSCLC celllines (Table 5). All primary tumors and 14 NSCLC cell lines wereanalyzed using an oligonucleotide array platform with a medianresolution of 50 kB (Brennan, C., et al. (2004) Cancer Res 64,4744-4748), and the remaining 20 NSCLC cell lines were interrogatedusing a cDNA array platform with a median resolution of 100 kB (Aguirre,A. J., et al. (2004) Proc Natl Acad Sci USA 101, 9067-9072). Eleven celllines were analyzed by both platforms, revealing a high level ofconcordance between the two datasets (correlation coefficient between0.88-0.95). Additionally, as previously demonstrated (Brennan, C., etal. (2004) Cancer Res 64, 4744-4748), the higher resolution of theoligonucleotide array platform relative to cDNA arrays uncovered severaladditional highly focal CNAs and greater structural detail of each CNA(FIG. 7).

To identify copy number changes above noise background, a modifiedversion of the circular binary segmentation methodology as describedpreviously was applied (Aguirre, A. J., et al. (2004) Proc Natl Acad SciUSA 101, 9067-9072; Olshen, A. B., et al. (2004) Biostatistics 5,557-572) (see also Materials and Methods). The most striking feature ofthe NSCLC dataset was the large number of CNAs (n=334) which, along withthe high degree of structural complexity for each CNA, promptedfiltering of the dataset through an algorithm proven effective indefining and prioritizing CNAs across large highly complex array-CGHdatasets (Aguirre, A. J., et al. (2004) Proc Natl Acad Sci USA 101,9067-9072). This algorithm applies several criteria including (i)recurrence of high-threshold amplification or deletion (above the 97thpercentile or below the 3rd percentile) in at least two specimens, (ii)presence of a high threshold event in at least one primary tumorspecimen, (iii) statistically significant median aberration, and (iv) apeak intensity equal to or greater than absolute Log₂ value of 0.8 ineither a cell line or primary tumor (i.e., beyond 0.5% quintiles). Froma collection of 334 CNAs, this algorithm yielded 126 so-called‘high-confidence’ Minimal Common Regions (MCRs) that satisfied at leastthree of the four criteria (Table 7). Ninety-one of these MCRs wereamplifications and 35 were deletions. Although the median size of the126 high-confidence MCRs defined automatically by the algorithm wascalculated to be 1.42 Mb, 23% (29) of these MCRs spanned 0.5 Mb or lesswith a median of 4 genes (Table 7; Table 7 shows high-confidence MCRs inlung adenocarcinoma and squamous cell carcinoma, spanning less than 1MB). The numbers of primary tumors (T) or cell lines (C) with gain orloss, and amplification or deletion, are listed, respectively. The MCRat 11q11 has recently been shown by Sebat et al. (Sebat, et al. (2004)Science 305:525) to be a copy number polymorphism (ORF511, chromosome11q11). Moreover, given that the parameters used by the automated MCRdefinition program are relatively conservative and the fact that not allgenes are represented by probes on these microarrays, real-time QPCRmeasurement of gene dosage and FISH analysis can further refine andnarrow the boundaries of an MCR, as illustrated by the 8p12-p11.2amplicon (Table 7 and FIG. 4), thereby presenting a large number of lociwith a highly tractable number of candidate cancer genes for furtheranalysis.

On first analysis, the dataset contained all the regional gains andlosses previously identified in NSCLC by chromosomal CGH andconventional genomic methodologies including the known gains at 1q31,3q25-27, 5p13-14, and 8q23-24 as well as the known deletions at 3p,8p22, 9p21-22, 13q22, and 17p12-13 (FIG. 1). Furthermore, virtually allof the genes implicated in the pathogenesis of NSCLC were containedwithin the high-confidence MCRs including p16^(INK4A) and RB1 tumorsuppressor genes, and MYC, EGFR and KRAS2 oncogenes (Table 7). The wholeshort arm of chromosome 3 was consistently lost, with a peak recurrenceat around 50 MB (29 out of 79 samples, 36%). Segmentation did notidentify obvious homozygous deletions which would point to a specifictarget in this recurrently lost region of 3p, however this regioncontains RASSF1, TUSC2, SEMA3B and FHIT, genes that have been previouslyshown to have LOH in NSCLC (Balsara and Testa, (2002 Oncogene 21:6877)(FIG. 1 and FIG. 8). The confidence level ascribed to these 126 selectedMCRs was further supported by verification with real-time QPCR on 17randomly selected MCRs.

Common and Disease-Specific Genomic Features

Primary NSCLC consists of two major subtypes, the squamous cellcarcinoma and the adenocarcinoma subtypes. A comparison of overall copynumber variation for high-intensity events across the genomes of thesetwo subtypes revealed a higher degree of genomic complexity in thesquamous cell carcinoma subtype (FIG. 2). Additionally, further analysisrevealed that squamous carcinoma samples harbor more complexrearrangements with multiple short segments linked together whencompared to the adenocarcinoma subset.

However, despite this qualitative difference in their genomic profiles,the most remarkable aspect of the subtype comparison was the significantoverlap of CNAs between the two primary tumor datasets, includinggains/amplifications at 1q, 5p, and 20q and losses/deletions at 3p and9p. To identify recurrent gained/amplified or lost/deleted regions thatwere distinct for each histological dataset, the incidence of events ineach primary tumor group were compared and significance estimated bypermutation testing (see Materials and Methods). Strikingly, these twogroups presented very few unique changes compared to within-groupvariation. In particular, two regions on the long arm of chromosome 3,from 3q11.2 to 3q13.2 and from 3q26.3 to 3q29, were selectively gainedand/or amplified in lung squamous cell carcinoma. These findings are inagreement with previous reports showing that the only genomic regionconsistently differentiating squamous cell carcinomas versusadenocarcinoma is a gain/amplification of the telomeric part ofchromosome 3q in squamous carcinoma (Balsara and Testa, 2002; Bjorkqvistet al., 1998; Luk et al., 2001; Pei et al., 2001; Petersen et al.,1997). In addition, gains/amplifications on the long arm of chromosome22, from 22q11 to 22q12.2 were selectively present in lung squamous cellcarcinoma, a region not previously implicated. The analysis did notreveal any genomic regions of loss or deletion that could differentiatethe lung squamous cell carcinoma from adenocarcinoma in the presentdataset. In addition, the analysis did not identify any genomic regionsthat were unique for the lung adenocarcinoma.

Given the remarkable similarity between the patterns of CNAs in the twocell types, it was determined what part of the difference in expressionprofiling reported between squamous cell carcinoma and adenocarcinomawas related to genes residing within chromosome 3q and 22q. Using at-test, probes that most significantly differentiated the twohistological subgroups in the expression profiling datasets wereidentified. The probes corresponding to genes previously reported asable to differentiate lung squamous cell carcinoma from adenocarcinomawere included, including p63, several members of the keratin family,collagen type VII, α 1 and desmoglein 3 (Bhattacharjee, A., et al.(2001) Proc Natl Acad Sci USA 98, 13790-13795; Garber, M. E., et al.(2001) Proc Natl Acad Sci USA 98, 13784-13789). Most of the genes thatsignificantly differentiated squamous cell carcinoma from adenocarcinomashowed sharp upregulation in the squamous cell carcinomas and no changeor modest downregulation in the adenocarcinomas. When the probes thatshowed a significant difference between the two subsets were mapped totheir genomic position, only two chromosomal arms, the long arms ofchromosome 3 and 22 showed a statistically significant enrichment inmapped probes, compared to that expected by chance alone. The sameanalysis was performed with another recently published lung cancerexpression profiling dataset (Bhattacharjee, A., et al. (2001) Proc NatlAcad Sci USA 98, 13790-13795). Only chromosome 3q, but not 22q, showed astatistically significant enrichment in the number of probes that wereoverexpressed in lung squamous cell carcinoma as compared to theadenocarcinomas. These findings suggest that genes residing within 3qand possibly 22q often show copy-number driven expression in squamouscell carcinomas. Gains/amplification of 3q is an event that is frequent,but not always present in squamous cell carcinomas, both in the presentdataset, where it was present in 54% of the samples, as well as in theliterature (where the reported frequencies are between 50 and 85% of thecases) (Balsara, B. R., and Testa, J. R. (2002) Oncogene 21, 6877-6883;Bjorkqvist, A. M., et al. (1998) Genes Chromosomes Cancer 22, 79-82;Luk, C., et al. (2001) Cancer Genet Cytogenet 125, 87-99; Pei, J., et al(2001) Genes Chromosomes Cancer 31, 282-287; Petersen, I., et al. (1997)Cancer Res 57, 2331-2335). Therefore, it was determined that squamouscell carcinomas without 3q gains/amplification nevertheless showedoverexpression of genes mapping to 3q. When additive tree-baseddendrograms were generated using only the genes located on 3q, most ofthe squamous samples clustered together, irrespectively of the presenceor absence of gains of 3q (FIG. 3). These findings suggest that genedosage amplification or transcriptional deregulation of one or, morelikely, several genes present in 3q is important in driving the cellularsquamous phenotype. Interestingly, using this selected population of 3qand 22q genes, three squamous cell carcinoma samples, S1, S5 and S14,did not cluster together with the other squamous cell carcinoma samplesin both 3q and 22q dendrograms (FIG. 3), but rather clustered theadenocarcinomas, suggesting that there is at least one different pathwaydriving the squamous cell carcinoma phenotype in a subgroup of squamouscell carcinomas, not related to genes residing in 3q or 22q.

Because oncogenes are usually associated with high-level amplificationsand tumor suppression genes are often defined by narrow homozygousdeletions, the presence of high-intensity events, it was determinedwhether these events were selectively present in one dataset but not inthe other. All the amplifications and deletions present in 15% or moreof the adenocarcinoma samples were present in at least one of thesquamous call carcinoma samples except for the amplification on the longarm of chromosome 8. Conversely, with two exceptions (see below), all ofthe recurrent (>15%) squamous cell carcinoma amplifications anddeletions were present in the adenocarcinoma tumors, again suggestingthe high degree of similarity between squamous cell carcinoma andadenocarcinomas even for high-intensity events.

As noted above, there were two highly recurrent (>15%) amplificationsthat were found to be present only in the squamous cell carcinomadataset, presenting the possibility of uncovering subtype-specific CNAsof pathobiological importance. The first region mapped to the long armof chromosome 3 (3q26-3qter). The second squamous cellcarcinoma-specific amplicon located at 8p12-p11.2 was a novel ampliconand was subjected to detailed structural analysis.

Common and Distinct Genomic Features in AC and SCC

Previous analyses of the NSCLC genome using low-resolution chromosomalor BAC array CGH have consistently shown that the only region of thegenome differentiating SCCs from ACs was 3q26-q29 (Bjorkqvist, A. et al.(1998) Genes Chromosomes Cancer 22, 79-82; Luk, C. et al. (2001) CancerGenet Cytogenet 125, 87-99; Pei, J. et al. (2001) Genes ChromosomesCancer 31, 282-7; Petersen, I. et al. (1997) Cancer Res 57, 2331-5;Balsara, B. R. & Testa, J. R. (2002) Oncogene 21, 6877-83). With thishigher-resolution platform capable of detecting previously unrecognizedfocal CNAs (see above), it was sought to determine whether there existadditional genomic events that are characteristic of either SCC or AC.Surprisingly, the genomic profiles of AC and SCC were highly overlappingsuch that neither supervised nor unsupervised clustering of the globalCGH profiles was able to classify these tumors according to theirhistopathological subtypes (FIG. 1 and data not shown). Next, it wasasked whether there are significant regional differences between AC andSCC subtypes. To this end, a permutation test was designed to comparethe incidence of events in each primary tumor subtypes and to estimatethe significance (see Materials and Methods). This permutation testidentified only one region of gain/amplification on the long arm ofchromosome 3, from 180 Mb to 199 Mb, corresponding to 3q26 to 3q29, thatwas significantly targeted in the SSCs (FIG. 3 a). Therefore, despitestrikingly distinct histological presentations, SCC and AC areremarkably similar on the genomic level and are likely driven by many ofthe same oncogenes and tumor suppressor gene mutations.

It was further hypothesized that the defined MCR on 3q harbors gene(s)driving the SCC phenotype and such resident target(s) can beup-regulated by mechanisms other than gene amplification since, althoughcommon among SCC, gain/amplification of 3q from 180-199 Mb is notpresent in all cases of SCC (54% in our samples and between 50 and 85%in the literature (Bjorkqvist, A. et al. (1998) Genes Chromosomes Cancer22, 79-82; Luk, C. et al. (2001) Cancer Genet Cytogenet 125, 87-99; Pei,J. et al. (2001) Genes Chromosomes Cancer 31, 282-7; Petersen, I. et al.(1997) Cancer Res 57, 2331-5; Balsara, B. R. & Testa, J. R. (2002)Oncogene 21, 6877-83). Thus, by comparing expression patterns of probesresiding within 3q180-199 Mb region, would be identified genes that areconsistently overexpressed in SCC versus AC regardless of copy numberstatus and such genes might have a higher probability to play a criticalin driving SCC phenotype. To this end, a one-way ANOVA and a post-hocBonferroni test using all the probes (n=166, corresponding to 106 genes)residing within the 180 to 199 Mb boundaries on 3q (see Material andMethods) identified a subset of genes showing significant overexpressionin SCCs versus ACs, and were overexpressed even in the absence of genecopy number gains on 3q in SCCs: p63, Claudin 1, Phosphatidylinositolglycan, class X, and discs large homolog 1 (Table 8). The p63 gene ismost notable given its seminal role in squamous tissue development andits links to squamous cancer subtypes (McKeon, F. (2004) Genes Dev 18,465-9; Massion, P. P. et al. (2003) Cancer Res 63, 7113-21; Westfall, M.D. & Pietenpol, J. A. (2004) Carcinogenesis 25, 857-64; Koster, M. I. etal. (2004) Genes Dev 18, 126-31).

To further corroborate the above finding, the global expression profiledata was analyzed for the same NSCLC samples using SAM (SignificantAnalysis for Microarray Data, (Tusher, V. G., Tibshirani, R. & Chu, G.(2001) Proc Natl Acad Sci USA 98, 5116-21)). A total of 297 probes werefound to be significantly different between SCCs and ACs, based on aq-value (false discovery rate, FDR) cutoff of 0.05 (Storey, J. D. &Tibshirani, R. (2003) Proc Natl Acad Sci USA 100, 9440-5). Of this listof differentially expressed genes, it was whether their differentialexpressions was driven by underlying copy number events. To this end,all of the 297 differentially expressed probes were mapped to theircorresponding genomic positions and, using a 10 Mb moving windowanalysis across the genome and then applying a Fisher's exact test (seeMaterials and Methods), those genomic regions that were significantlyenriched for differentially expressed probes were identified. As shownin FIG. 3 b, this global analysis again identified 3q 180-199 MB as thegenomic region whose resident genes are most significantly enriched foramong the list of differentially expressed genes between AC and SCC.Similar results were also obtained when a published lung cancerexpression profile dataset was utilized (Bhattacharjee, A. et al. (2001)Proc Natl Acad Sci USA 98, 13790-5) (data not shown). In conclusion, theintegrated aCGH and expression analyses strongly implicate a limitednumber of genes residing within 3q as key drivers of SCChistopathological phenotype.

Characterization of Squamous Cell Carcinoma Subtype Specific Amplicon on8p12-p11.2

The limited resolution of the microarrays and the conservativeparameters of the automated algorithm in defining MCRs prompted adetailed mapping of the 8p12-p11.2 amplicon by real-time QPCR to betterdefine the event boundaries. Primer pairs were designed for genesresiding around and within the putative amplicon and the correspondinggene dosages were determined by QPCR in multiple primary tumor and cellline samples (FIG. 4A). Based on two informative primary tumor cases,PT3 and PT5, this analysis defined the MCR as 0.14 MB in size andcontaining 2 annotated genes (FIG. 4A).

As shown in FIG. 4A, QPCR analysis clearly positioned the FGFR1 geneoutside of the telomeric boundary of this MCR. Since FGFR1 has beenconsidered to be a prime candidate for this 8p amplicon in other cancertypes (Edwards, J., et al. (2003) Clin Cancer Res 9, 5271-5281; Mao, X.,et al. (2003) J Invest Dermatol 121, 618-627; Simon, R., et al. (2001)Cancer Res 61, 4514-4519), interphase fluorescence in situ hybridization(FISH) was conducted to verify the definition of this boundary. UsingBAC #1 (spanning part of the amplicon and not including FGFR1) as aprobe, FISH analysis of all 8 samples (two cell lines and six primarytumors) confirmed the presence of a high-copy number amplicon at8p12-p11.2 (FIGS. 4A-F, FIGS. 9-11). Consistent with the QPCR data, FISHanalysis with BAC #2 (outside of the MCR and including FGFR1) on PT3revealed only two copies (FIGS. 4A and 4D), proving unequivocally thatFGFR1 is not amplified in this sample. FISH analysis with BAC #3 definedthe telomeric boundaries in PT5, again in accordance with aCGH and QPCRdata (FIGS. 4E and F).

Since copy number aberrations are believed to be one of the mechanismsfor altered gene expression, integration of DNA copy number and RNAexpression data can serve as an effective early filter for cullingbystanders from true targets. To this end, the expression pattern ofgenes residing within this MCR was assessed by both gene expressionprofiling and by directed RT-QPCR (FIG. 5). RAB11FIP1 and FGFR1 wereincluded in these analyses as controls outside of the centromeric andtelomeric boundaries of the MCR, respectively. Specifically, thetarget(s) should be overexpressed relative to normal, and suchoverexpression should exhibit copy number driven patterns, namely,overexpression observed in samples harboring the amplicon in question.As shown in FIG. 5, only WHSC1L1 exhibited patterns of expressionconsistent with that of a candidate target of amplification. Incontrast, LETM2 represented a probable bystander based on inconsistentcopy number driven overexpression as compared to normal and tumorsamples without the amplification. Both RAB11FIP1 and FGFR1 showed anexpression pattern that was consistent with their placement outside ofthe amplicon MCR.

Comparison of the Lung CNAs with Pancreatic CNAs.

The very large number of amplifications in human lung cancer presentssignificant challenges in prioritizing those amplified oncogenes forfurther validation. Given the common and overlapping role of many bonafide oncogenes or tumor suppressor genes across multiple cancer types,the assumption that the comparison with array-CGH datasets from othercancer types could identify common CNAs harboring highly importantoncogenes or tumor suppressor genes was tested. To this end, the NSCLCgenome was compared with that of pancreatic ductal adenocarcinoma (PA)genome (Aguirre, A. J., et al. (2004) Proc Natl Acad Sci USA 101,9067-9072). These comparisons identified several loci that are known tobe commonly targeted in these cancers including KRAS, c-MYC andINK^(4a/ARF) loci, as well as other novel loci. Among these, one locuson 20q11 (position 29.66-29.88 MB) was amplified in one pancreatic cellline as well as in one lung adenocarcinoma primary tumor and in 4 lungcell lines. Overlap of the aCGH profiles permitted delimitation of anMCR of 220 kB in size (position 29.66 to 29.88 MB) containing 5 genes,ID1, COX412, BCL2L1, TPX2 and MYLK2. Among these, only BCL2L1, a knownoncogene implicated in multiple cancer types and TPX2 showed copy-drivenexpression (FIG. 6). Interestingly, TPX2 showed high-level expression inmost lung cell lines and primary tumors tested, when compared withnormal controls (FIG. 6). These findings suggest that, in addition toBCL2L1 in this locus, TPX2 also plays a role in lung and pancreatictumorigenesis.

As described herein, the NSCLC genome is highly complex and rearrangedwith many previously unrecognized regions of amplification and deletion,all with potential pathogenetic relevance in this lethal disease. Inparticular, 126 high-confidence CNAs have been identified and defined.It is notable that more than 20% of these (29 of the 126) span less than0.5 MB with a median of 4 resident genes. The significance of thesefindings rests on the fact that, to date, only a limited number of geneshave been shown to be critical for the pathogenesis and maintenance ofnon-small cell lung cancer (Minna, J. D., et al. (2002) Cancer Cell 1,49-52).

As an estimate of the likelihood of discovering novel NSCLC genes fromthis dataset, the list of genes residing within the high-confident MCRswas compared with the presumed cancer-relevant tyrosine kinases(Bardelli, A., et al. (2003) Science 300, 949), phosphatases (Wang, Z.,et al. (2004) Science 304, 1164-1166), the phosphatidylinositol3-kinases (PI3Ks) (Samuels, Y., et al. (2004) Science 304 554), as wellas the current census of cancer genes compiled by Futreal, et al.((2004) Nat Rev Cancer 4, 177-183). While many implicated cancer genesindeed map to larger CNAs, none of these known cancer genes residewithin the coordinates of focal MCRs of 0.5 MB or less (Table 3). Thus,it can be concluded that genes residing within the high-confidence MCRsidentified here represent candidates from which novel lung canceroncogenes and tumor suppressor genes will emerge. Additionally, whilenone of the proviral common integration sites mapped to any of the focalMCRs, several of the known microRNAs (miRNAs) were located within theMCRs (Table 7). The role of microRNA in cancer has been recentlyestablished and the presence of miRNAs within MCRs suggest that they mayplay a role in carcinogenes in lung cancer (Calin, G. A., et al. (2004a)Proc Natl Acad Sci USA 101, 11755-11760; Calin, G. A., et al. (2004b)Proc Natl Acad Sci USA 101, 2999-3004; Metzler, M., et al. (2004) GenesChromosomes Cancer 39, 167-169).

Employing custom bioinformatic tools with clinical histopathologicalcorrelation, the present high resolution genome-wide analysis has alsouncovered a difference in overall genomic complexity between the twomajor subtypes of NSCLC. Specifically, the squamous cell carcinomasubtype harbors genomic features suggestive of a higher degree ofgenomic instability compared to the adenocarcinoma subtype, consistentwith previous reports (Massion, P. P., et al. (2002) Cancer Res 62,3636-3640). While the basis for this difference is not clear, it ispossible that this higher complexity relates to tobacco smoke exposure.Epidemiological studies have shown that lung squamous carcinomas aremore causally linked to tobacco smoke than other lung cancer subtypes.Correspondingly, cytogenetic analyses have shown that adenocarcinomasarising in smokers tend to exhibit higher frequency of chromosomalrearrangements than those in non-smokers (Sanchez-Cespedes, M., et al.(2001) Cancer Res 61, 1309-1313). Taken together, these observationsraise the possibility that tobacco smoke may target pathways governinggenome stability and/or checkpoint control. In addition to genotoxicproperties of its carcinogens, tobacco may also promote localinflammation leading to increased lung epithelial cell renewal, therebyaccelerating telomere erosion and eventual entry into crisis, a triadknown to engender complex genomic rearrangements (Chin, K., et al.(2004) Nat Genet. 36, 984-988; Kinouchi, Y., et al. (1998) JGastroenterol 33, 343-348; O'Sullivan, J. N., et al. (2002) Nat Genet.32, 280-284; Romanov, S. R., et al. (2001) Nature 409, 633-637), as wellas epithelial carcinogenesis (Artandi, S. E., et al. (2000) Nature 406,641-645).

However, irrespective of the mechanistic differences in their evolution,it is most remarkable that the vast majority of CNAs are shared betweenboth squamous cell carcinoma and adenocarcinoma subtypes, an observationpointing to common pathogenetic events driving the transformation oflung epithelial cells. The results presented herein clearly demonstratethat the genome of the lung squamous cell carcinoma possess more focalCNAs than that of the lung adenocarcinomas. Yet, it is striking thatdespite these wide spread genomic differences only two chromosomalregions are consistently different between these two histologicalphenotypes. While gain at 22q has not been previously reported assquamous cell carcinoma specific, gains at the telomeric region onchromosome 3q have been consistently, albeit not uniquely, associatedwith the squamous cell carcinoma subtype (Balsara, B. R., and Testa, J.R. (2002) Oncogene 21, 6877-6883; Bjorkqvist, A. M., et al. (1998) GenesChromosomes Cancer 22, 79-82; Luk, C., et al. (2001) Cancer GenetCytogenet 125, 87-99; Pei, J., et al. (2001) Genes Chromosomes Cancer31, 282-287; Petersen, I., et al. (1997) Cancer Res 57, 2331-2335). Ofpotential significance is the observation that this region has beenreported to be the most common and early genetic alteration in squamouscell carcinomas of the head and neck, a group of tumors that sharesimilar developmental, histological and pathogenetic features withsquamous cell carcinomas of the lung (Huang, Q., et al. (2002) GenesChromosomes Cancer 34, 224-233).

One potential explanation for the similarity between squamous cellcarcinomas and adenocarcinomas is that these two histological types oflung cancer are derived from the same lung stem/precursor cell and thatonly a few number of unique genetic alterations are sufficient to conferto these cells either an adenocarcinoma or a squamous cell carcinomaphenotype. Indeed, there is some experimental evidence that the alveolartype II cell is a pluripotential stem cell involved in the genesis ofhuman adenocarcinoma and squamous cell carcinoma (Ten Have-Opbroek, A.A., et al. (1997) Histol Histopathol 12, 319-336). Additionally,approximately 2% of lung cancers contain both adeno as well as squamouspopulations of cells—adenosquamous carcinomas (Nakagawa, K., et al.(2003) Ann Thorac Surg 75, 1740-1744; Sridhar, K. S., et al. (1992) Am JClin Oncol 15, 356-362). Interestingly, it has been shown by genetic andimmunohistochemical analysis, that the two populations within each tumorare monoclonal (Kanazawa, H., et al. (2000) Am J Pathol 156, 1289-1298).The data presented herein show that overexpression of genes residingwithin 3q and possibly 22q, either mediated by amplification or by othermechanisms, could selectively induce lung squamous cell carcinomaphenotypes upon a genetic background that is common betweenadenocarcinomas and squamous cell carcinomas. Interestingly, among thegenes that did show overexpression in squamous samples irrespectively ofcopy number changes was p63. Overexpression of selective splice variantsof p63 has been reported in several squamous carcinomas (Massion, P. P.,et al. (2003) Cancer Res 63, 7113-7121; McKeon, F. (2004) Genes Dev 18,465-469) and mutations in p63 has been reported in several human geneticdisorders affecting ectodermal development (Westfall, M. D., andPietenpol, J. A. (2004) Carcinogenesis 25, 857-864). p63 null-mice showprofound defects or frank loss of the entire spectrum of epithelialtissues (Mills, A. A., et al. (1999) Nature 398, 708-713; Yang, A., etal. (1999) Nature 398, 714-718). Conditional transgenic mice expressingp63 isoforms in the epithelial cells lining the bronchioles of the lungdeveloped severe squamous metaplasia (Koster, M. I., et al. (2004) GenesDev 18, 126-131). Together with these findings, other experimental datasupport the view that p63 exerts a critical role in maintaining theproliferative capacity of epidermal cell populations as well as drivingan epithelial stratification program (McKeon, F. (2004) Genes Dev 18,465-469). Other genes overexpressed in 3q irrespective of copy numberchanges have been implicated in epithelial development and squamous cellcarcinomas, such as for example Cystatin A (Ebert, E., et al (1997) AdvExp Med Biol 421, 259-265; Pernu, H., et al. (1990) Acta Histochem 88,53-57) and RP42 (or SCRO, squamous cell carcinoma-related oncogene)(Estilo, C. L., et al. (2003) Clin Cancer Res 9, 2300-2306). Based onthese data and on the prerogatives of the genes overexpressed in 3q,both adenocarcinomas and squamous cell carcinomas could potentiallyarise from a common origin, possibly a stem cell. Dysregulation ofgenes, as for examples genes residing within 3q, could then drive asquamous cell phenotype upon a common tumorigenic background.

Because oncogenes are usually associated with high-level amplificationsand tumor suppression genes are often defined by narrow homozygousdeletions, it was determined whether the presence of high-intensityevents were selectively present in one dataset but not in the other.Besides the amplification on 3q, a second amplicon associated with thesquamous cell carcinoma subtype mapped to chromosome 8p12-p11.2 spanninga region of 1.48 MB. Although novel for NSCLC, this amplicon has beenobserved in several other cancer types including breast, prostate,bladder carcinomas and T-cell lymphomas (Dib, A., et al. (1995) Oncogene10, 995-1001; Edwards, J., et al. (2003) Clin Cancer Res 9, 5271-5281;Mao, X., et al. (2003) J Invest Dermatol 121, 618-627; Ray, M. E., etal. (2004) Cancer Res 64, 40-47; Simon, R., et al. (2001) Cancer Res 61,4514-4519). While FGFR1 resides within this larger CNA and has beenconsidered the prime candidate target of this amplification, detailedQPCR and FISH mapping performed in this study defined both boundariesand narrowed the minimal common region, excluding the FGFR1 gene.Correspondingly, expression analysis demonstrated that FGFR1 was notoverexpressed in 4 out of 5 tumors that harbor this amplicon andsequence analysis of exons encoding the juxtamembrane and kinase domainsof FGFR1 failed to reveal any mutations. These findings in lung cancerare consistent with recent studies in breast cancer showing that FGFR1does not play a pathogenetic role in breast cancer cells harboringamplification of this 8p locus (Dib, A., et al. (1995) Oncogene 10,995-1001; Ray, M. E., et al. (2004) Cancer Res 64, 40-47), therebypointing to other resident gene(s) as the true target of this 8p12-p11.2amplicon such as, for example, the genes showing activity in the tumorcell growth assays.

Gene dosage alterations represent a common mechanism of oncogeneactivation and tumor suppressor inactivation by modulating expression oftheir target genes. Thus, integration of DNA copy number with expressionprovides a powerful early filter for culling bystanders of CNAs. Inother words, it is a requirement that the true target of a CNA exhibitappropriate altered expression in a copy number driven manner. However,definition of over- or under-expressed levels is a challenge in itself,particularly in cases where the true cell-of-origin remains unknown andthus a premalignant physiological frame-of-reference is not available.In this study, there are 3 independent normal RNA references, isolatedfrom adjacent histologically normal lung tissues. Using such areference, one can eliminate LETM2 (leucine zipper-EF-hand containingtransmembrane protein 2), since it is not overexpressed in severalsamples showing amplification. This gene has extensive sequencesimilarity with LETM1, that encodes a putative member of the EF-handfamily of Ca(2+)-binding proteins (Endele, S., et al. (1999) Genomics60, 218-225). These proteins contain two EF-hands, a transmembranedomain, a leucine zipper, and several coiled-coil domains. LETM1 isintegral of the inner mitochondrial membrane and is involved in both K+homeostasis and organelle volume control (Nowikovsky, K., et al. (2004)J Biol Chem 279, 30307-30315). Haploinsufficiency of LETM1 has beenimplicated in the neuromuscular features of WHS patients.

By EST database searching with mouse Nsd1 and utilizing human WHSC1 asprobe to screen an amnion cell cDNA library, Angrand et al. ((2001)Genomics 74, 79-88) cloned a partial sequence for WHSC1L1. The deduced1,437-amino acid protein contains 2 PWWP domains involved inprotein-protein interaction, 5 PHD-type zinc finger motifs found inchromatin-associated proteins, a SAC (SET-associated cys-rich) domain, aSET domain, and a C-terminal C5HCH domain. They also identified avariant, arising from alternative polyadenylation and exon splicing,that encodes a deduced 645-amino acid peptide that contains a singlePWWP domain. WHSC1L1 shares 68% and 55% identity with mouse Nsd1 andhuman NSD1 respectively, over a 700-amino acid block containing the SAC,SET, and C5HCH domains. Amplification of WHSC1L1 has been demonstratedin breast cancer (Angrand, P. O., et al. (2001) Genomics 74, 79-88).Additionally, this gene is involved in a chromosomal translocation, t(8;11)(p11.2; p15), along with NUP98, that preserves all the domains inWHSC1L1 excluding one PWWP domain (Rosati, R., et al. (2002) Blood 99,3857-3860). This gene belongs to the SET2 family of histone lysinemethyltransferases (Schneider, R., et al. (2002) Trends Biochem Sci 27,396-402). Two other genes within this family present extensive sequencesimilarity with WHSC1L1, NSD1 and WHSC1, and both of them have beenimplicated in cancer. NSD1 coregulates and transactivates the androgenreceptor in prostate cancer cells (Wang, X., et al. (2001) J Biol Chem276, 40417-40423). Additionally, it is fused to the nucleoporin geneNUP98 in the recurrent chromosomal translocation t(5; 11)(q35; p15.5) inde novo childhood acute myeloid leukemia (Jaju, R. J., et al. (2001)Blood 98, 1264-1267). WHSC1 is involved in a frequent chromosomaltranslocation, t(4;14)(p16.3;q32.3), in Multiple Myeloma (Stec, I., etal. (1998) Hum Mol Genet. 7, 1071-1082).

The high number of CNAs identified in NSCLC represents a challenge inprioritizing and selecting MCRs for detailed analysis. As many of thealready described oncogenes and tumors suppressor genes are involved intumorigenesis across different tissue types, it was reasoned thatmapping overlapping MCR across aCGH datasets of different cancer typesmight yield novel loci that harbor additional genes essential fortumorigenesis. Indeed, when the recently published pancreatic collectionof MCRs was compared (Aguirre, A. J., et al. (2004) Proc Natl Acad SciUSA 101, 9067-9072) with the adenocarcinoma and squamous cell carcinomaCNAs, CNAs including known genes already implicated in both cancers,such as KRAS, c-MYC and INK^(4a)/ARF were identified. Additionally othernovel loci in common between the two datasets were uncovered. Amongthem, a MCR in chromosome 20 was present in the high-confidence list inboth pancreas and lung cancer and contained 5 genes, of which only twoshowed copy-number driven expression (FIG. 6). The identification ofBCL2L1 within a focal amplicon shared between NSCLC and pancreaticadenocarcinoma suggests that one of the mechanisms of BCL2L1 activationin lung cancer is via chromosomal amplification, and confirms a criticaloncogenic role in pancreatic adenocarcinoma for BCL2L1, as previouslysuggested (Bold, R. J., et al. (2001) Cancer 92, 1122-1129; Trauzold,A., et al. (2003) Br J Cancer 89, 1714-1721; Xu, Z., et al. (2001) Int JCancer 94, 268-274). More surprising is the involvement of TPX2 in thishighly recurrent amplicon. TPX2 showed high level of expression not onlyin the amplified samples but also in samples not showing genome dosagechanges. TPX2 is required for targeting Aurora-A kinase to the spindleapparatus. Elevated expression of Aurora-A kinase have been reported inbreast, bladder, colon, ovarian and pancreatic cancers and correlateswith chromosomal instability and clinically aggressive disease. Up to62% of breast cancers overexpress Aurora-A kinase, even where geneamplification is not detected. Elevated Aurora-A kinase expressioncauses abnormalities in mitosis and chromosome segregation andectopically expressed Aurora-A kinase can transform rodent cells (Anand,S., et al. (2003) Cancer Cell 3, 51-62; Bischoff, J. R., et al. (1998)Embo J 17, 3052-3065; Miyoshi, Y., et al. (2001) Int J Cancer 92,370-373). However, no reports of specific amplification oroverexpression of Aurora-A kinase have been reported in NSCLC. Indeed inthe present dataset no amplification of Aurora Kinase A was alsoevident. TPX2 activates Aurora-A kinase (Gruss, O. J., et al. (2002) NatCell Biol 4, 871-879) and when overexpressed, it induces accumulation ofcells in G2-M phase and polyploidization (Heidebrecht, H. J., et al.(2003) Mol Cancer Res 1, 271-279). Manda et al. ((1999) Genomics 61,5-14) have demonstrated that this gene is overexpressed in lung cancertissue when compared with normal lung and its expression tightlycorrelates with poor prognosis in patients with neuroblastoma (Krams,M., et al. (2003) J Clin Oncol 21, 1810-1818). Additionally, usingOncomine (Rhodes, D. R., et al. (2004) Neoplasia 6, 1-6), the expressionlevel of TPX2 in different cancer types was compared to thecorresponding levels in normal tissues. Lung squamous cell carcinoma andadenocarcinoma, lung small cell carcinoma (Bhattacharjee, A., et al.(2001) Proc Natl Acad Sci USA 98, 13790-13795; Garber, M. E., et al.(2001) Proc Natl Acad Sci USA 98, 13784-13789), as well as prostate(Dhanasekaran, S. M., et al. (2001) Nature 412, 822-826; LaTulippe, E.,et al. (2002) Cancer Res 62, 4499-4506) and hepatocellular carcinoma(Chen, X., et al. (2002) Mol Biol Cell 13, 1929-1939) showed significantoverexpression of TPX2 when compared with the respective normal tissues.Intriguingly, in the present expression dataset, a high correlationexisted between TPX2 and Aurora-A kinase expression (r=0.7801, P<0.001)and correlated even more with many other genes involved in spindleformation and mitotic progression as for example, Bub1 (r=0.93,P<0.001), CDC20 (r=0.93, P<0.001), Aurora-B kinase (r=0.90, P<0.001).The amplification of TPX2 and the correlation of its expression withgenes involved in spindle formation and progression through the cellcycle suggest a possible critical role for TPX2 in lung and pancreaticcarcinogenesis.

Cross Tumor Type Genomic Comparison

The remarkable degree of overlap between lung cancer subtypes promptedcomparison with the recently generated high-resolution genomic profileof another lethal epithelial tumor type, pancreatic adenocarcinoma(PDAC). Although the majority of the defined CNAs are distinct betweenlung and PDAC, the intersection between these two datasets did reveal 20shared novel loci, in addition to expected common genomic alterationssuch as KRAS, c-MYC and INK4a/ARF (Table 3). Thus, cross tumor typecomparison holds the potential to serve as filter for CNAs that harbortargets that are potentially relevant to multiple cancer types.

One of the shared loci was a focal amplification at 8p12-p11.2 (position37.84-39.72 MB) encompassing FGFR1, a cancer-relevant gene notpreviously implicated in lung cancer. Detailed mapping of the 8p12-p11.2amplicon by real-time quantitative PCR (QPCR) narrowed the MCR to 0.14MB in size, as defined by two informative primary tumor cases, PT3 andPT5 (FIG. 4 a and data not shown). As QPCR analysis clearly positionedthe FGFR1 gene outside of the telomeric boundary, this focal MCRcontained only 2 annotated genes: WHSC1L1 and LETM2 (FIG. 4 a). Sinceprevious studies have implicated FGFR1 as the prime target of 8pamplicon in other cancer types (Simon, R. et al. (2001) Cancer Res 61,4514-9), interphase FISH was performed to verify the amplicon boundariesin several informative samples. FISH on the six primary tumors and on 2cell lines showing the amplification confirmed presence of a high-copynumber amplicon at 8p (FIG. 4 a-b, FIGS. 9-12). Consistent with QPCRdata, FISH with a BAC outside of the MCR and including FGFR1 on PT3revealed only two copies (FIGS. 12 a and 12 d), providing clear evidencethat FGFR1 is not amplified in this sample. The integration of DNA copynumber and RNA expression data was used to cull bystanders from truetargets of the amplicon (Pollack, J. R. et al. (2002) Proc Natl Acad SciUSA 99, 12963-8). Both gene expression profiling and RT-QPCR (FIG. 5 anddata not shown) demonstrated consistent overexpression of WHSC1L1. Theother MCR resident gene, LETM2, did not show consistent overexpressionin the presence of gene amplification. RAB11FIP1 and FGFR1, two genespositioned external to the telomeric and centromeric boundaries of theMCR, respectively showed an expression pattern that was consistent withtheir placement outside of the amplicon MCR.

To further evaluate the relevance of FGFR1 versus WHSC1L1 in lungcancer, the biological impact of siRNA-mediated knockdown of each targetwas assessed in cell lines with and without 8p amplification (NCI-H1703and NCI-H1395 respectively). For all genes, RT-QPCR documented >70%knock-down following SMART siRNA pool transduction (data not shown). Insoft agar assays with NCI-H1703, siRNA-mediated knock-down of WHSC1L1resulted in 50% reduction in the number of H1703 soft agar colonies,while near complete FGFR1 depletion had no impact on colony formation insoft agar. As expected, knock-down of these two genes had no effect onNCI-H1395 colony formation. The copy number-driven expression data,coupled with knockdown studies, argues against a role for FGFR1 in lungcancers harboring an 8p amplicon and points to WUSC1L1 as a potentialtarget of this amplification event.

Another amplicon shared in the NSCLC and PDAC datasets mapped to 20q11.2harboring BCL2L1 (previously BCL-xL), a known oncogene implicated inmultiple cancer types. This amplicon was detected in one primary lungAC, one adenosquamous cell line, one SCC cell line and 2 AC cell lines.These samples together delimited the MCR to 220 kB, spanning position29.66 to 29.88 Mb and containing 5 genes: ID1, COX4I2, BCL2L1, TPX2 andMYLK2. Although BCL2L1 indeed exhibited modestly elevated expressionTPX2 was the only gene showing high-level copy number-driven expressionin most lung cell lines and primary tumors tested, when compared withRNA derived from normal lung (FIG. 4). These findings suggest that, inaddition to a known oncogene BCL2L1, TPX2 is a potential candidateoncogenes targeted for amplification in both lung and pancreas cancers.

Example II

From an initial list of 1848 genes that are amplified in lung cancers, acommon set of genes that are amplified in both lung cancers andpancreatic ductal adenocarcinoma based on array CGH analyses wereselected. These genes were further selected for their presence in mostof the lung cancer primary tumor samples, as resulted from affymetrixexpression profile analyses. Affymetrix expression profiles of lungcancer samples were analyzed using the mas5 P/M/A call algorithmimplemented in Bioconductor open source project. The gene list was thenfiltered for targets for either antibody or small molecule therapeuticagents. Genes encoding known transmembrane proteins or secreted proteinswere categorized as potential targets for antibody therapy. Genes withproducts containing in silico predicted transmembrane domains or signalpeptide were also categorized as potential targets for antibody therapyunless other evidence indicated that the gene products were not on thecell surface or were extracellular. Known G-protein coupled receptors,enzymes, ion-channels, nuclear receptors, as well as genes encodingproteins containing in silico predicted functional domains for the aboveclasses were categorized as potential targets for small moleculetherapeutics. The filtered gene list as described herein is shown inTable 9A. Also included in Table 9 are eleven genes that were selectedfrom LU smallest amplicons (0.5 kb or less) (see Table 9B).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

TABLE 1 Markers of the Invention which reside in MCRs of deletion anddisplay decreased expression. Gene (start of Pos (Mb) (end SymbolChromosome gene) of gene) Locus Link MCR ID LOC219431 11 5516257855175891 GeneID: 219431 LDLU_11_55.096877_55.175716 OR4C11 11 5512749355128425 GeneID: 219429 LDLU_11_55.096877_55.175716 OR4C16 11 5509618055097112 GeneID: 219428 LDLU_11_55.096877_55.175716 HSMPP8 13 1910588019144657 GeneID: 54737 LDLU_13_19.14411_19.558476 LOC387901 13 1942944919430578 GeneID: 387901 LDLU_13_19.14411_19.558476 LOC390377 13 1929027419290720 GeneID: 390377 LDLU_13_19.14411_19.558476 LOC400099 13 1933606519337976 GeneID: 400099 LDLU_13_19.14411_19.558476 PSPC1 13 1914752819255082 GeneID: 55269 LDLU_13_19.14411_19.558476 ZNF198 13 1943081019558939 GeneID: 7750 LDLU_13_19.14411_19.558476 ZNF237 13 1930960519335664 GeneID: 9205 LDLU_13_19.14411_19.558476 FARP1 13 9766347097898828 GeneID: 10160 LDLU_13_97.474214_97.898599 KPNB3 13 9740437497474551 GeneID: 3843 LDLU_13_97.474214_97.898599 ZNF183L1 13 9762604097627522 GeneID: 140432 LDLU_13_97.474214_97.898599 BAGE3 21 1004271310080194 GeneID: 85318 LDLU_21_9.932177_10.079854 HCP41 21 99578469958038 GeneID: 360186 LDLU_21_9.932177_10.079854 TPTE 21 992807510012791 GeneID: 7179 LDLU_21_9.932177_10.079854 CASP2 7 142502239142521622 GeneID: 835 LDLU_7_142.276497_142.97 CLCN1 7 142530056142565934 GeneID: 1180 LDLU_7_142.276497_142.97 CTAGE6 7 142889833142892437 GeneID: 340307 LDLU_7_142.276497_142.97 EPHA1 7 142605042142622822 GeneID: 2041 LDLU_7_142.276497_142.97 FLJ40722 7 142835382142859822 GeneID: 285966 LDLU_7_142.276497_142.97 FLJ90586 7 142498900142501978 GeneID: 135932 LDLU_7_142.276497_142.97 GSTK1 7 142477401142483043 GeneID: 373156 LDLU_7_142.276497_142.97 KIAA0773 7 142567332142575732 GeneID: 9715 LDLU_7_142.276497_142.97 LOC154761 7 142963860142968610 GeneID: 154761 LDLU_7_142.276497_142.97 LOC202775 7 142593623142595003 GeneID: 202775 LDLU_7_142.276497_142.97 LOC346521 7 142526611142526991 GeneID: 346521 LDLU_7_142.276497_142.97 LOC441294 7 142785710142788317 GeneID: 441294 LDLU_7_142.276497_142.97 OR10AC1P 7 142724887142725841 GeneID: 392133 LDLU_7_142.276497_142.97 OR2R1P 7 142702395142703341 GeneID: 392132 LDLU_7_142.276497_142.97 OR6W1P 7 142276218142277719 GeneID: 89883 LDLU_7_142.276497_142.97 PIP 7 142346011142353671 GeneID: 5304 LDLU_7_142.276497_142.97 TAS2R39 7 142397349142398362 GeneID: 259285 LDLU_7_142.276497_142.97 TAS2R40 7 142436009142436980 GeneID: 259286 LDLU_7_142.276497_142.97 TAS2R41 7 142691803142692724 GeneID: 259287 LDLU_7_142.276497_142.97 TAS2R60 7 142657383142658339 GeneID: 338398 LDLU_7_142.276497_142.97 TAS2R62P 7 142650965142651903 GeneID: 338399 LDLU_7_142.276497_142.97 ZYX 7 142595233142605041 GeneID: 7791 LDLU_7_142.276497_142.97

TABLE 2 Markers of the Invention which reside in MCRs of amplificationand display increased expression. Gene Pos (Mb) (start Pos (Mb) (endSymbol Chromosome of gene) of gene) Locus Link MCR ID FLJ13941 1 22848582355155 GeneID: 79906 LALU_1_2.350016_2.468358 KIAA0450 1 24426912469131 GeneID: 9651 LALU_1_2.350016_2.468358 PEX10 1 2368405 2376172GeneID: 5192 LALU_1_2.350016_2.468358 RER1 1 2355491 2367352 GeneID:11079 LALU_1_2.350016_2.468358 CAMK1G 1 206145440 206175679 GeneID:57172 LALU_1_206.174776_206.237991 G0S2 1 206237160 206238128 GeneID:50486 LALU_1_206.174776_206.237991 LAMB3 1 206176640 206214152 GeneID:3914 LALU_1_206.174776_206.237991 CDCA8 1 37827246 37844482 GeneID:55143 LALU_1_37.817059_38.132489 FHL3 1 38131536 38134200 GeneID: 2275LALU_1_37.817059_38.132489 FLJ20508 1 37816336 37825285 GeneID: 54955LALU_1_37.817059_38.132489 FLJ23476 1 37937707 37942939 GeneID: 79693LALU_1_37.817059_38.132489 FLJ31434 1 37930253 37936313 GeneID: 149175LALU_1_37.817059_38.132489 FLJ33655 1 37895051 37899882 GeneID: 284656LALU_1_37.817059_38.132489 FLJ45459 1 37943062 37944219 GeneID: 127687LALU_1_37.817059_38.132489 INPP5B 1 37996852 38086883 GeneID: 3633LALU_1_37.817059_38.132489 LOC391026 1 37901200 37914277 GeneID: 391026LALU_1_37.817059_38.132489 LOC440579 1 37826195 37827002 GeneID: 440579LALU_1_37.817059_38.132489 LOC440580 1 37848654 37891456 GeneID: 440580LALU_1_37.817059_38.132489 LOC440581 1 37917400 37929374 GeneID: 440581LALU_1_37.817059_38.132489 MTF1 1 37948944 37992474 GeneID: 4520LALU_1_37.817059_38.132489 SF3A3 1 38091740 38124738 GeneID: 10946LALU_1_37.817059_38.132489 C10orf94 10 135256286 135271757 GeneID: 93426LALU_10_135.2356235_135.275316 CYP2E1 10 135229746 135241501 GeneID:1571 LALU_10_135.2356235_135.275316 FLJ44653 10 135269237 135272343GeneID: 399833 LALU_10_135.2356235_135.275316 BLNK 10 97941447 98021316GeneID: 29760 LALU_10_97.622871_98.114607 C10orf130 10 97749873 97769126GeneID: 387707 LALU_10_97.622871_98.114607 CCNJ 10 97793141 97810612GeneID: 54619 LALU_10_97.622871_98.114607 DNTT 10 98054202 98088311GeneID: 1791 LALU_10_97.622871_98.114607 FLJ34077 10 97624802 97657532GeneID: 404033 LALU_10_97.622871_98.114607 LOC387706 10 9769971597717198 GeneID: 387706 LALU_10_97.622871_198.114607 LOC399804 1097939099 97940396 GeneID: 399804 LALU_10_97.622871_98.114607 TLL2 1098114356 98263658 GeneID: 7093 LALU_10_97.622871_98.114607 TMEM10 1098092969 98109049 GeneID: 93377 LALU_10_97.622871_98.114607 ZNF518 1097879646 97910520 GeneID: 9849 LALU_10_97.622871_98.114607 ERBB3 1254760154 54782854 GeneID: 2065 LALU_12_54.760154_54.840103 FLJ14451 1254798297 54802545 GeneID: 84872 LALU_12_54.760154_54.840103 MBC2 1254808321 54824722 GeneID: 23344 LALU_12_54.760154_54.840103 MLC1SA 1254833923 54838027 GeneID: 140465 LALU_12_54.760154_54.840103 MYL6 1254838413 54841627 GeneID: 4637 LALU_12_54.760154_54.840103 PA2G4 1254784692 54793353 GeneID: 5036 LALU_12_54.760154_54.840103 RPL41 1254796641 54797883 GeneID: 6171 LALU_12_54.760154_54.840103 FAM14A 1493663874 93665676 GeneID: 83982 LALU_14_93.663981_94.033875 KIAA1622 1493710402 93815825 GeneID: 57718 LALU_14_93.663981_94.033875 SERPINA1 1493914463 93924877 GeneID: 5265 LALU_14_93.663981_94.033875 SERPINA10 1493819405 93829114 GeneID: 51156 LALU_14_93.663981_94.033875 SERPINA11 1493978556 93998476 GeneID: 256394 LALU_14_93.663981_94.033875 SERPINA1214 94023374 94053934 GeneID: 145264 LALU_14_93.663981_94.033875 SERPINA214 93900404 93914178 GeneID: 390502 LALU_14_93.663981_94.033875 SERPINA614 93840339 93859426 GeneID: 866 LALU_14_93.663981_94.033875 SERPINA9 1494000716 94005949 GeneID: 327657 LALU_14_93.663981_94.033875 FLJ11171 1669873706 69881013 GeneID: 55783 LALU_16_69.618966_69.899852 HYDIN 1669618512 69822070 GeneID: 54768 LALU_16_69.618966_69.899852 DSC1 1826963214 26996817 GeneID: 1823 LALU_18_26.828128_27.310282 DSC2 1826900005 26936375 GeneID: 1824 LALU_18_26.828128_27.310282 DSC3 1826825029 26876687 GeneID: 1825 LALU_18_26.828128_27.310282 DSG1 1827152050 27190457 GeneID: 1828 LALU_18_26.828128_27.310282 DSG3 1827281756 27310474 GeneID: 1830 LALU_18_26.828128_27.310282 DSG4 1827210738 27247877 GeneID: 147409 LALU_18_26.828128_27.310282 KCNC3 1955510577 55524446 GeneID: 3748 LALU_19_55.518384_55.678241 LOC284361 1955670405 55678420 GeneID: 284361 LALU_19_55.518384_55.678241 MYBPC2 1955628004 55661390 GeneID: 4606 LALU_19_55.518384_55.678241 NAPSA 1955553547 55560743 GeneID: 9476 LALU_19_55.518384_55.678241 NAPSB 1955528856 55539830 GeneID: 256236 LALU_19_55.518384_55.678241 NR1H2 1955571550 55578051 GeneID: 7376 LALU_19_55.518384_55.678241 POLD1 1955579408 55613082 GeneID: 5424 LALU_19_55.518384_55.678241 SPIB 1955614027 55624058 GeneID: 6689 LALU_19_55.518384_55.678241 BBS5 2170161522 170188672 GeneID: 129880 LALU_2_170.186554_170.319334 FLJ219012 170211776 170255829 GeneID: 79675 LALU_2_170.186554_170.319334 KBTBD102 170191719 170208279 GeneID: 10324 LALU_2_170.186554_170.319334 PPIG 2170266357 170319761 GeneID: 9360 LALU_2_170.186554_170.319334 ANKRD23 296925526 96931632 GeneID: 200539 LALU_2_96.681846_96.952466 CNNM3 296903865 96921508 GeneID: 26505 LALU_2_96.681846_96.952466 CNNM4 296848754 96899485 GeneID: 26504 LALU_2_96.681846_96.952466 FLJ10081 296680785 96725957 GeneID: 55683 LALU_2_96.681846_96.952466 LMAN2L 296793540 96827675 GeneID: 81562 LALU_2_96.681846_96.952466 LOC442030 296730448 96739640 GeneID: 442030 LALU_2_96.681846_96.952466 LOC90342 296757829 96792496 GeneID: 90342 LALU_2_96.681846_96.952466 MGC41816 296935654 96945622 GeneID: 51239 LALU_2_96.681846_96.952466 SEMA4C 296947350 96957582 GeneID: 54910 LALU_2_96.681846_96.952466 BCL2L1 2029715924 29774317 GeneID: 598 LALU_20_29.6573635_29.878599 COX4I2 2029689352 29696461 GeneID: 84701 LALU_20_29.6573635_29.878599 ID1 2029656753 29657974 GeneID: 3397 LALU_20_29.6573635_29.878599 MYLK2 2029871045 29886153 GeneID: 85366 LALU_20_29.6573635_29.878599 TPX2 2029790565 29853264 GeneID: 22974 LALU_20_29.6573635_29.878599 BRD9 5916856 943161 GeneID: 65980 LALU_5_0.532951_0.917447 FLJ10565 5 665461706664 GeneID: 55722 LALU_5_0.532951_0.917447 LOC442127 5 732027 747916GeneID: 442127 LALU_5_0.532951_0.917447 SLC9A3 5 526425 577447 GeneID:6550 LALU_5_0.532951_0.917447 TPPP 5 713891 731181 GeneID: 11076LALU_5_0.532951_0.917447 ZDHHC11 5 848907 904101 GeneID: 79844LALU_5_0.532951_0.917447 PCDHB10 5 140552310 140555393 GeneID: 56126LALU_5_140.545848_140.662462 PCDHB11 5 140559532 140562802 GeneID: 56125LALU_5_140.545848_140.662462 PCDHB12 5 140568664 140571866 GeneID: 56124LALU_5_140.545848_140.662462 PCDHB13 5 140573693 140577177 GeneID: 56123LALU_5_140.545848_140.662462 PCDHB14 5 140583262 140586044 GeneID: 56122LALU_5_140.545848_140.662462 PCDHB15 5 140605331 140607986 GeneID: 56121LALU_5_140.545848_140.662462 PCDHB16 5 140541164 140545980 GeneID: 57717LALU_5_140.545848_140.662462 PCDHB18 5 140594307 140596906 GeneID: 54660LALU_5_140.545848_140.662462 PCDHB19P 5 140599675 140607986 GeneID:84054 LALU_5_140.545848_140.662462 PCDHB9 5 140547077 140551295 GeneID:56127 LALU_5_140.545848_140.662462 SLC25A2 5 140662380 140663616 GeneID:83884 LALU_5_140.545848_140.662462 FGFR1 8 38389449 38445293 GeneID:2260 LALU_8_38.241102_38.45 HTPAP 8 38241417 38245872 GeneID: 84513LALU_8_38.241102_38.45 LETM2 8 38363177 38385218 GeneID: 137994LALU_8_38.241102_38.45 LOC441345 8 38358653 38360217 GeneID: 441345LALU_8_38.241102_38.45 WHSC1L1 8 38251717 38358947 GeneID: 54904LALU_8_38.241102_38.45

TABLE 3 High-confident focal MCRs MCR Recurrence Minimal Common Regions(MCRs) Gain/Loss Position Size Max/Min Cell Amp/Del Cytogenetic Band(MB) (MB) Value # Transcripts % Tumors lines Tumors Cell linesCandidates Gains/Amplification 1p36.32-1p36.32 2.37-2.47 0.10 2.92 2 229 9 3 0 PEX10, RER1 1p34.3-1p34.3 37.82-38.13 0.32 2.16 11 18 7 8 1 1FLJ31434, YRDC, FLJ45459 1q32.2-1q32.2 206.17-206.24 0.06 1.14 3 39 1517 1 4 LAMB3 2q11.2-2q11.2 96.68-96.95 0.27 1.39 8 35 14 16 4 4 CNNM3,CNNM4, SEMA4C 2q31.1-2q31.1 170.19-170.32 0.13 2.67 4 28 11 13 1 1 PPIG5p15.33-5p15.33 0.53-0.92 0.38 1.99 5 60 27 24 6 15 BRD9 5q31.3-5q31.3140.55-140.66 0.12 1.59 11 19 6 10 2 4 PCDHB11, PCDHB12, PCDHB138p12-8p11.22*  38.24-38.45* 0.21 1.98 2 35 17 12 6 4 FGFR1, WHSC1L1,LETM2 10q24.1-10q24.1 97.62-98.11 0.49 1.96 8 16 3 10 1 1 CCNJ10q26.3-10q26.3 135.24-135.24 0.01 1.04 1 11 1 8 1 3 C10orf9412q13.2-12q13.2 54.72-54.84 0.12 1.98 8 29 10 14 2 2 ERBB314q32.13-14q32.13 93.66-94.03 0.37 1.15 9 29 6 18 1 4 KIAA162216q22.2-16q22.2 69.62-69.87 0.26 1.4 2 18 6 9 1 4 Hs.36878118q12.1-18q12.1 26.83-27.31 0.48 1.1 6 19 9 7 2 1 DSC1, DSC2, DSG219q13.33-19q13.33 55.52-55.68 0.16 0.73 8 17 5 9 1 1 SPIB20q11.21-20q11.21 29.66-29.88 0.22 1.08 5 42 13 23 1 8 BCL2L1, TPX2Loss/Deletion 7q34-7q34 142.28-142.44 0.16 −1.39 4 13 7 4 2 1 LOC15476111q11-11q11{circumflex over ( )}  55.1-55.18 0.08 −4.06 6 23 13 6 7 1OR4C11, OR4C6, OR4V1P{circumflex over ( )} 13q12.11-13q12.11 19.14-19.560.41 −3.51 2 34 14 14 1 7 HSMPP8, PSPC1 13q32.2-13q32.2 97.47-97.9  0.42−3.25 6 29 13 11 1 8 FARP1 21p11.2-21p11.1  9.93-10.08 0.15 −1.33 5 17 96 1 1 TPTE

TABLE 4 Cell line description. Name ATCC# Diagnosis Age/Race/Sex NCI-H522 CRL 5810 Adenocarcinoma 60/White/Male NCI-H1437 CRL 5872 Pleuraleffusion adenocarcinoma 60/White/Male NCI-H1703 CRL 5889 Adenosquamouscarcinoma 54/White/Male NCI-H1975 CRL 5908 Adenocarcinoma NA/NA/FemaleHs 618.T CRL 7380 Adenocarcinoma 60/White/Female NCI-H 647 CRL 5834Pleural effusion adenosquamous 56/White/Male carcinoma NCI-H1781 CRL5894 Pleural effusion adenocarcinoma 66/White/Female NCI-H1838 CRL 5899Adenocarcinoma NA/NA/Female NCI-H 2347 CRL 5942 Adenocarcinoma54/White/Female NCI-H 2122 CRL 5985 Pleural effusion adenocarcinoma46/White/Female NCI-H23 CRL 5800 Adenocarcinoma 51/Black/Male NCI-H1299CRL 5803 Lymph node carcinoma 43/White/Male NCI-H 358 CRL 5807Bronchoalveolar carcinoma NA/NA/Male NCI-H 838 CRL 5844 Lymph nodeadenocarcinoma 59/White/Male SK-LU-1 HTB 57 Adenocarcinoma60/White/Female NCI-H 292 CRL 1848 Mucoepidermoid pulmonary carcinoma32/Black/Female NCI-H1373 CRL 5866 Adenocarcinoma 56/Black/Male NCI-H1395 CRL 5868 Adenocarcinoma 55/White/Female NCI-H1650 CRL 5883 Pleuraleffusion adenocarcinoma 27/White/Male H 1944 CRL 5907 Soft-tissueadenocarcinoma 62/White/Female NCI-H1568 CRL 5876 Lymph nodeadenocarcinoma 48/White/Female NCI-H1993 CRL 5909 Lymph nodeadenocarcinoma 47/White/Female NCI-H 2170 CRL 5928 Squamous cellcarcinoma NA/NA/Male ChaGo-K-1 HTB 168 Bronchogenic carcinoma 45/NA/MaleNCI-H 460 HTB 177 Pleural effusion carcinoma NA/NA/Male NCI-H 596 HTB178 Adenosquamous carcinoma 73/White/Male NCI-H 520 HTB 182 Squamouscell carcinoma NA/NA/Male NCI-H 2030 CRL 5914 Lymph node adenocarcinomaNA/NA/Male NCI-H 1435 CRL 5870 Adenocarcinoma 35/NA/Female NCI-H 1573CRL 5877 Soft tissue adenocarcinoma 35/White/Female NCI-H 1623 CRL 5881Lymph node adenocarcinoma 58/White/Male NCI-H 1648 CRL 5882 Lymph nodeadenocarcinoma 39/Black/Male NCI-H 3255 Adenocarcinoma 47/White/FemaleDFCI.PT Adenocarcinoma 54/White/Male

TABLE 5 Diagnosis Cellularity (%) Age/Race/Sex Samples obtained from theCooperative Human Tissue Network (CHTN) Consortium Poorly differentiatedadenocarcinoma >70 83/Black/Female Adenocarcinoma >70 55/White/FemaleMetastatic adenocarcinoma >70 73/Black/Female Adenocarcinoma >7066/B/Male Squamous cell carcinoma >70 74/White/Male Poorlydifferentiated squamous carcinoma >70 72/Asian/Male Poorlydifferentiated squamous carcinoma >70 56/Black/Male Adenocarcinoma >7076/White/Male Squamous cell carcinoma >70 68/White/Female Moderatelydifferentiated adenocarcinoma >70 71/White/Female Moderatelydifferentiated adenocarcinoma >70 78/White/Male Adenocarcinoma >7046/NA/Male Moderately differentiated squamous cell carcinoma >7071/White/Male Squamous cell carcinoma >70 47/White/Male Squamous cellcarcinoma >70 67/White/Male Squamous cell carcinoma >70 67/White/MaleAdenocarcinoma >70 76/White/Male Samples obtained from Brigham and WomenHospital tumor bank. Moderately differentiated squamous carcinoma >7070/NA/Male Poorly differentiated squamous carcinoma >70 57/NA/MaleModerately differentiated squamous carcinoma >70 58/NA/Male Moderatelydifferentiated squamous carcinoma >70 74/NA/Female Poorly differentiatedadenocarcinoma >70 66/NA/Male Poorly differentiated adenocarcinoma >7088/NA/Male Poorly differentiated adenocarcinoma >70 79/NA/MaleModerately differentiated squamous carcinoma >70 70/NA/Male Moderatelydifferentiated squamous carcinoma >70 70/NA/Male Moderately/Poorly(mixed) differentiated adenocarcinoma >70 50/NA/Female Mixeddifferentiated adenocarcinoma >70 65/NA/Female Moderately differentiatedadenocarcinoma >70 49/NA/Female Moderately/Poorly (mixed) differentiatedadenocarcinoma >70 59/NA/Male Moderately/Poorly (mixed) differentiatedadenocarcinoma >70 53/NA/Female Poorly differentiated adenocarcinoma >7066/NA/Male Moderately differentiated squamous carcinoma >70 74/NA/FemalePoorly differentiated squamous carcinoma >70 69/NA/Male Poorlydifferentiated adenocarcinoma >70 72/NA/Male Moderately differentiatedsquamous carcinoma >70 64/NA/Male Poorly differentiated squamouscarcinoma >70 49/NA/Male Poorly differentiated squamous carcinoma >7074/NA/Female Well differentiated squamous carcinoma >70 51/NA/MalePoorly differentiated squamous carcinoma >70 60/NA/Male Moderatelydifferentiated squamous carcinoma >70 68/NA/Male Moderately/Poorlydifferentiated squamous carcinoma >70 76/NA/Male Moderatelydifferentiated squamous carcinoma >70 53/NA/Female Squamouscarcinoma >70 77/NA/Female Moderately differentiated squamouscarcinoma >70 66/NA/Male

TABLE 6 Oligos DNA only,RNA   PCR, RT PCR,  Primer name Forward SequenceReverse Sequence Project only or both Sequencing 80223_NorthernCCGCTCTAATGTCTGCATCA CAAAGAGGTTCAGGGAGCTG Lung RNA only Northern probes(SEQ ID NO: 341) (SEQ ID NO: 542) 9070_northern TCGAGCTCCCCATTAAAGAAGCCCATGTCACTCATAGGG Lung RNA only Northern probes (SEQ ID NO: 342)(SEQ ID NO: 543) 137362_northern CTTCCAGGACATGGGCTTTAACCAGGTATTCCACCTGCTG Lung RNA only Northern probes (SEQ ID NO: 343)(SEQ ID NO: 544) 155_northern TGACCAACGTGTTCGTGACT AGAGTGAAGGTGCCCATGATLung RNA only Northern probes (SEQ ID NO: 344) (SEQ ID NO: 545)6770_northern TAGCGACATTCAAGCTGTGC GAGGTCGATGCTGAGTAGCC Lung RNA onlyNorthern probes (SEQ ID NO: 345) (SEQ ID NO: 546) 9530_northernGGAGCAGCCACCATATCCTA CTGATGGACACTGCAAGGAA Lung RNA only Northern probes(SEQ ID NO: 346) (SEQ ID NO: 547) 23259_northern GATTTCCAACGTGCAAAGGTCAAAGACCCCAGTGGTGAGT Lung RNA only Northern probes (SEQ ID NO: 347)(SEQ ID NO: 548) 2260_northern CGATGTGCAGAGCATCAACT TCGATGTGCTTTAGCCACTGLung RNA only Northern probes (SEQ ID NO: 348) (SEQ ID NO: 549)27257_Northern AGGAGGGAAGAGGTTTTGGA GCCACAGTGATGTGTGAAGG Lung RNA onlyNorthern probes (SEQ ID NO: 349) (SEQ ID NO: 550) 1978_NorthernCGGGGACTACAGCACGAC GGCGAAGGTGGCTTTTATTT Lung RNA only Northern probes(SEQ ID NO: 350) (SEQ ID NO: 551) 84513_Northern GAGGAGATGTGGCTCTACCGTCAGGTTGCTTTCTGAACACC Lung RNA only Northern probes (SEQ ID NO: 351)(SEQ ID NO: 552) 22974_Northern CAGGCTACGGAGAGAACTGGGGTGCAGTTGGTGAGGTTTT Lung RNA only Northern probes (SEQ ID NO: 352)(SEQ ID NO: 553) 598_Northern TCTGGTCCCTTGCAGCTAGT GCTGAGGCCATAAACAGCTCLung RNA only Northern probes (SEQ ID NO: 353) (SEQ ID NO: 554)LETM2_north CAAGGAGGAACAGAGCCAAG ATTTGTGATTTGGCCTGGAG Lung RNA onlyNorthern probes (SEQ ID NO: 354) (SEQ ID NO: 555) W_Northern_bothGGTGCCCGAGAATATCATGT GTCTTGCCCCCTGACACTTA Lung RNA only Northern probes(SEQ ID NO: 355) (SEQ ID NO: 556) W_Northern_short CAGAATGCTACAGGGGATGGTGGTGGTTTTTAGGCTGGAC Lung RNA only Northern probes (SEQ ID NO: 356)(SEQ ID NO: 557) W_Northern_long ATGAAAACCCTTGTGGCTTGTCTTCATGCATCTGCTTTGG Lung RNA only Northern probes  (SEQ ID NO: 357)(SEQ ID NO: 558) 11102_CDS4_1 CCCACTAATTTCCAGCCATC TGTGAAGGCCCTCCTAAGTTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 358) (SEQ ID NO: 559)within exon 116135_CDS1_1 ATCATGAGACAGCCCACAAC GTGACCAGCATGGCATAATC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 359) (SEQ ID NO: 560)within exon 1230_CDS1_1 TCATGACCAACAAAGGCAAT ATGGGAATTCACTCACCACA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 360) (SEQ ID NO: 561)within exon 128853_CDS4_1 GCAGCTTCTTCTGTGTCTCTTT ACCTGGTGCCTTAGTCCTTGLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 361) (SEQ ID NO: 562)within exon 151648_CDS4_1 GCTGGGCTTGCTTTATTCTT CACCTGAAACTCAGCAGTCACLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 362) (SEQ ID NO: 563)within exon 1605_CDS0_1 CATTGCTCCTCCAACAGAGA CAGCTTCTGACACCCGAGTA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 363) (SEQ ID NO: 564)within exon 1776_CDS1_1 TCCATGGCATTCTTGTCTTC TGCTTCTCCTCCTCTCCATC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 364) (SEQ ID NO: 565)within exon 201595_CDS01 ACCGAACACGTAATGCTGAG CTCCCTGTTATCAGGTGCTTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 365) (SEQ ID NO: 566)within exon 2177_CDS21-1 GGAGAGCACAGCAGATGAGA CAAAGTTCTGCTCCACCAAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 366) (SEQ ID NO: 567)within exon 3921_W_GENE_1 TTCTGGGTGAGTTCCGTGTA CTGACCGGCTTTCATCACTA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 367) (SEQ ID NO: 568)within exon 4336_CDS0_1 CTCCGAACACTTCAGCATACA GCAGATCCAGTCCTCCTCTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 368) (SEQ ID NO: 569)within exon 4968_CDS12_1 GGCCCAGATAAAGGTCATGT AGTCCAAGTCCCAGTGTGTG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 369) (SEQ ID NO: 570)within exon 51066_CDS4_1 GAGTCATGGATTCCACCAGA GAAGATGGCCGACACTCTCT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 370) (SEQ ID NO: 571)within exon 5289_CDS13_1 CCACGTTAGGAGCAACCTTT ATGACATCCAAGGAAGCAGA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 371) (SEQ ID NO: 572)within exon 57406_CDS0_1 GGCCTGCAGTACTCAACTGAC GGAGCAGAGCTGAAGCATTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 372) (SEQ ID NO: 573)within exon 6091_CDS11_1 TCTCCTCTTTCATATCCTCCAAG CCCAGTTCCTCAAGCTCAATLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 373) (SEQ ID NO: 574)within exon 9390_CDS0_1 CAAATCAGTGCCCTCAGAGA TGCTGATGGTCCAGCTCTTA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 374) (SEQ ID NO: 575)within exon 2248_CDS0_1 TTCACAGACACGTACCACAGTCT TCCACGAGCTGGGCTATAATLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 375) (SEQ ID NO: 576)within exon 2249_CDS1_1 AGGAAGTGGGTGACCTTCAT GTGCACGTTCAAGGAGATTC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 376) (SEQ ID NO: 577)within exon 2263_CDS1_1 CTGTCAACATGCAGAGTGAA CACCACGGACAAAGAGATTG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 377) (SEQ ID NO: 578)within exon 9965_CDS2_1 ATGGGCAGGAAATGAGAGAG CAATGTGTACCGATCCGAGA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 378) (SEQ ID NO: 579)within exon 10077_W_GENE_1 CACCACCCAAGATCCTCTCT AGCTCAGGAGACCAGCATTTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 379) (SEQ ID NO: 580)within exon 1073_CDS1_1 CGATACAGCGAAGGAGTCTAAC TTTCAGGGATGGGACAACTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 380) (SEQ ID NO: 581)within exon 2064_CDS1_1 TGACACAGCTTATGCCCTATG GCAATCTGCATACACCAGTTC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 381) (SEQ ID NO: 582)within exon 26136_CDS3_1 TGCATCCACAGAGTGCTTTC TGCCCTCCTCCTAAGACATC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 382) (SEQ ID NO: 583)within exon 6540_CDS11_1 TTGAGGGTTCCGAGTCTGTAG ACTCCGCTGACCTACAACAA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 383) (SEQ ID NO: 584)within exon 65980_CDS10_1 ACACAGTCGGTGGCCTATTA GCTGCAGGCTAATCCTTTCT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 384) (SEQ ID NO: 585)within exon 836_CDS5_1 CCAGTGCGTATGGAGAAATG GCAATAAATGAATGGGCTGA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 385) (SEQ ID NO: 586)within exon 10204_CDS2_3 TTTCCTCCTCCCTCCTCTTC CATCCAAACATCATGCACAA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 386) (SEQ ID NO: 587)within exon 123228_CDS0_1 CTACTGCGGCAATCAGATGT GTGACTTCAGGGCTGATGAA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 387) (SEQ ID NO: 588)within exon 3911_CDS3_1 GGTGGAAGGGCCAAATATAA GGTGTCTATTTGGACTCTAAGCTCTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 388) (SEQ ID NO: 589)within exon 4649_CDS0_1 GCCAATGCATAGAATCCAAA AGCTGCATATCACCCAACAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 389) (SEQ ID NO: 590)within exon 56654_W_GENE_1 GAGACACAGAGACGCACACA GTTCCCAGAGGCGTACCTTALung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 390) (SEQ ID NO: 591)within exon 8065_COS0_1 TTGGAGGTAACCACGTTTCA TCAGCATCAGGGAGTTCAGTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 391) (SEQ ID NO: 592)within exon 2260_CDS0_1 TCTGGCTGTGGAAGTCACTC GGAGATCATCATCTATTGCACAGLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 392) (SEQ ID NO: 593)within exon 2037_CDS1_1 CATCTTGTACCACGGCAATG TGGTGTCATGGACCAAAGTC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 393) (SEQ ID NO: 594)within exon 6582_CDS1_1 GAATTCAACCAGGGCAGAGT GTTCACGAGCTCTGTGCTCTA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 394) (SEQ ID NO: 595)within exon 1230_CDS0_1 AACGGACAGCTTTGGATTTC ACCTCTTTGGGCTGGTATTG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 395) (SEQ ID NO: 596)within exon 1232_W_GENE_1 TGGAGGCGTAGTGGTAACAG ACCTGCTGTGGTGGTTTGTA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 396) (SEQ ID NO: 597)within exon 1605_CDS0_1 GGTGGTATCATCTGCCTGTG GTGCAGGAGGTCAATCCTTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 397) (SEQ ID NO: 598)within exon 84276_CDS2_1 CAGGCAGCAGAAGAGTTCAG TGGGACACTTGAGGAAGAAA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 398) (SEQ ID NO: 599)within exon 8927_CDS0_1 ATGGCAGAGCAGAACATCAG GTGTTATAGTTGGCTGGGCTCT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 399) (SEQ ID NO: 600)within exon 10324_CDS1_1 GGAAAGATCTGGCTCCAATG CAACTGAAGCTGAAAGACCATCLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 400) (SEQ ID NO: 601)within exon 114990_CDS1_1 GCCCTACATCTAAGCCAGAGA CGAGGTTGGGACTGAGAACTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 401) (SEQ ID NO: 602)within exon 1571_CDS2_1 GTCATAGCCGACATCCTCTTC TGCTGAGTAGGTGGAAGTTCTCLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 402) (SEQ ID NO: 603)within exon 1746_CDS0_1 GGGACGAACTGGTTCCATAG GCACATGGGTTCCTACCAGT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 403) (SEQ ID NO: 604)within exon 4661_CDS10_1 TTGGGTCAAATTGCTGAAGA ATCCTCTGGCTGGATGAAGA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 404) (SEQ ID NO: 605)within exon 51608_CDS0_1 CAAATAACCGGTCCAGGAGT CATGCACAACTGCGTATCAA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 405) (SEQ ID NO: 606)within exon 56121_W_GENE_1 ACGGGAGGCTCTGAAAGTAA ACGGTCGGAAAGCTACAGATLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 406) (SEQ ID NO: 607)within exon 57596_CDS2_1 AGAAGCTGTCGGCCTTGTAG ACCCTGCAGCTTCTCTGAAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 407) (SEQ ID NO: 608)within exon 84809_CDS4_1 TTCAAGGAACACCAGGATAGAA CCTCTCTCCACCGAGCTATCLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 408) (SEQ ID NO: 609)within exon 8837_CDS1_1 TCACCGACGAGTCTCAACTAA CGTGGTCCTTGTTGTCTCAG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 409) (SEQ ID NO: 610)within exon 9742_CDS12_1 ACTCCCTTCAAAGGAATGAATC GTGTTGGAATACGCGACAGALung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 410) (SEQ ID NO: 611)within exon 1978_EXON1 CAGCAGCTGCAGCCAGAC GGGGTGGTGCTGAAGAGC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 411) (SEQ ID NO: 612)within exon 55107_EXON2 CTTATCTGTGGACCCTGATGC GAGGGCCTCTTGTGATGGTA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 412) (SEQ ID NO: 613)within exon 5326_W_GENE_2 GGCTATCTGGAGCAGGAAAG TCCCTGGAGACCCAGATAAG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 413) (SEQ ID NO: 614)within exon 137362_CDS0_1 ATCATTCATCCAGGCCTCTC TGCCAAGCTCTGAGAAACTG LungRNA only Real-time PCR (SEQ ID NO: 414) (SEQ ID NO: 615) 137994_CDS0_1GGGATGAGATCACTGGGTCT GGCTTGGGCTTCACATCTAT Lung RNA only Real-time PCR(SEQ ID NO: 415) (SEQ ID NO: 616) 2249_CDS0_1 CCCAACAACTACAACGCCTACAGGAAGTGGGTGACCTTCAT Lung RNA only Real-time PCR (SEQ ID NO: 416)(SEQ ID NO: 617) 22941_CDS0_1U TCGAGACGACCAGCACTATC CATAAATGCTTGCCCATCTGLung RNA only Real-time PCR (SEQ ID NO: 417) (SEQ ID NO: 618)25960_CDS0_1 CTGGAGGGATTCCACTCATT TCAAAGCCACAGGGATGTAG Lung RNA onlyReal-time PCR (SEQ ID NO: 418) (SEQ ID NO: 619) 55107_CDS0_1AAGGACATCAGCCAGGAGAT TCCTTCTCCATCCAGGTTTC Lung RNA only Real-time PCR(SEQ ID NO: 419) (SEQ ID NO: 620) 6770_CDS0_1 CTGGCATGGACACAGACTTCTAGAGGGACTTCCAGCCAAC Lung RNA only Real-time PCR (SEQ ID NO: 420)(SEQ ID NO: 621) 80223_CDS0_1 ATCCCTAGCACGACACCTTC AAGAGGCGTCTTCTGCAAATLung RNA only Real-time PCR (SEQ ID NO: 421) (SEQ ID NO: 622)84513_CDS0_1 AGATGTGGCTCTACCGGAAC GGCTTGTCTGCTGTCTCTTG Lung RNA onlyReal-time PCR (SEQ ID NO: 422) (SEQ ID NO: 623) 8772_CDS0_1GAAGACCTGTGTGCAGCATT GCTGTCGATCTTGGTGTCTG Lung RNA only Real-time PCR(SEQ ID NO: 423) (SEQ ID NO: 624) 9070_CDS0_1 TGCCATCACAGTGGGAATACTTCGGATGTTCATCCTGTGT Lung RNA only Real-time PCR (SEQ ID NO: 424)(SEQ ID NO: 625) 9530_CDS0_1 GCACAACTTTCCTTGCAGTG TTGATTGTTGGGATGGTCACLung RNA only Real-time PCR (SEQ ID NO: 425) (SEQ ID NO: 626) 400841_DNAACAGGGGAGACAGAGCAAGA CTGGAAAAGCCACCTTTTTG Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 426) (SEQ ID NO: 627) within exon 155_CDS0_1GGAGCTCCAGGGTTCATAAG AAACAAACGGAGGGAAAGTG Lung RNA only Real-time PCR(SEQ ID NO: 427) (SEQ ID NO: 628) 1978_CDS0_1 CAGCCAGGCTTATGAAAGTGTGGAGGCACAAGGAGGTAT Lung RNA only Real-time PCR (SEQ ID NO: 428)(SEQ ID NO: 629) 220064_CDS0_1 TGCCTCCCGTGTGTTATTTA TTAGACCAGGCAATCCCTTCLung RNA only Real-Time PCR (SEQ ID NO: 429) (SEQ ID NO: 630)2248_CDS0_1 ATAACCTGGAGCCCTCTCAC GGCTCAAGGAGGAGAGTCAG Lung RNA onlyReal-time PCR (SEQ ID NO: 430) (SEQ ID NO: 631) 23259_CDS0_1GCCAAGTTGTGGTCTTCAAA AGGGCTATGCTGTCCATCTT Lung RNA only Real-time PCR(SEQ ID NO: 431) (SEQ ID NO: 632) 27257_CDS0_1 CGGTAGGAGGGAAGAGGTTTCATCTCGAAGCAGAACCAAG Lung RNA only Real-time PCR (SEQ ID NO: 432)(SEQ ID NO: 633) 11160_CDS0_1 GGCTCAGAGTGGTGGTTTCT GATGCTGAGTGGAACCAATGLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 433) (SEQ ID NO: 634)within exon 137362_CDS0_1 CTGGCTGCAGAAGAACTCAA GAGCAAGCAGATATTCCCATTTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 434) (SEQ ID NO: 635)within exon 137994_CDS2_1 CCCAGCACAAAGGAGATAGTT CAGGTTGTTGGTTCCAAATGLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 435) (SEQ ID NO: 636)within exon 22974_CDS0_1 GAGGGCCTTTCTGGTTCTCT TAGTCTGGCCTCCTCCAACT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 436) (SEQ ID NO: 637)within exon 25960_CDS16_1 CATCACTCTGGCCTCATCAC TGTCTGTGCAGCAAGCATAA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 437) (SEQ ID NO: 638)within exon 27257_CDS3_1 GTCTGTGATCAAATGCGTGA TCCATTCCTCGAGCAGATACT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 438) (SEQ ID NO: 639)within exon 54904_CDS0_2 CTCAGCCAGTAATTCTTCATACTGT CCCGAGAATATCATGTCCAGTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 439) (SEQ ID NO: 640)within exon 55290_CDS1_1 TCTTCACCAGTTCTGCCAAG GCACGCTGTTGTATGCAGAT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 440) (SEQ ID NO: 641)within exon 6770_CDS1_1 GCTGCTTGTTCTGTGGTGTT CTTGCTTTATGGGCTCAAGA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 441) (SEQ ID NO: 642)within exon 6867_CDS10_1 GGCAGTGATCTCCCAGATTT TCTTCCTCTGGCTCACCTTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 442) (SEQ ID NO: 643)within exon 80223_CDS0_1 CTCCTGCTCTCTGGCTTCTT CTATGACCCTGCCGTCCTAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 443) (SEQ ID NO: 644)within exon 84513_CDS4_1 TCATTGTCAGGTTGCTTTCTG TGGGCAGATGAATAGCAATAAGLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 444) (SEQ ID NO: 645)within exon 9070_CDS0_1 TGGAACACCCGTTTAACAAA CGTTCATACAAGCAGGCTCT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 445) (SEQ ID NO: 646)within exon 9530_CDS3_1 GCACAACTTTCCTTGCAGTG GATGGTCACTGGTGGCATAA LungDNA only Real-time PCR (SEQ ID NO: 446) (SEQ ID NO: 647) 23259TCCCTAGACGGACATTGAGG CAAAGACCCCAGTGGTGAGT Lung DNA only Real-time PCR(SEQ ID NO: 447) (SEQ ID NO: 648) 11212_CDS2_2 CTCGCAAGAACCATCTGACAGGGATCCTGTTTCCTTTCAA Lung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 448)(SEQ ID NO: 649) within exon 2017_CDS11_1 AGGCAGAGCTGAGCTACAGAGCCTCTGCGCTTTCATAGACA Lung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 449)(SEQ ID NO: 650) within exon 8500_CDS0_1 GTGCTTGTTTGGAGCAATGATGCACTGAAGTCGAAGGTTT Lung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 450)(SEQ ID NO: 651) within exon 8772_CDS1_1 ACACCAAGATCGACAGCATCTTGCGTTCTCCTTCTCTGTG Lung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 451)(SEQ ID NO: 652) within exon 55290_CDS0_1 CATCTGCACGCTGTTGTATGGCTGCTGCAATAGGTCTTCA Lung RNA only Real-time PCR (SEQ ID NO: 452)(SEQ ID NO: 653) 595_CDS0_1 TCCTGGTGAACAAGCTCAAG GTGTTTGCGGATGATCTGTTLung RNA only Real-time PCR (SEQ ID NO: 453) (SEQ ID NO: 654)9965_CDS0_1 CCACCAGGCTTCAGGAGTAG ACCGGGACAGCAAGTTATTC Lung RNA onlyReal-time PCR (SEQ ID NO: 454) (SEQ ID NO: 655) 116461_CDS2_1CCACTGCAGCTACCGTAGAA ACTGCAATGGGAAGACCAAT Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 455) (SEQ ID NO: 656) within exon126789_CDS4_1 GGTGAAGACGATTCTGGAGAG GTTCCCGTACAGCACTGACTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 456) (SEQ ID NO: 657)within exon 170575_CDS0_1 ACTTGGTTAAGGGAGGCTGA GCAAAGTTGCAAAGGTGATG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 457) (SEQ ID NO: 658)within exon 22823_CDS1_1 AGAAAGGACCAAATGCCAAA AACACTGCTGGACATTGGTTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 458) (SEQ ID NO: 659)within exon 3987_CDS7_1 AGCTGAGACCTTAGGAAGGAAAT TCCTCTTCTCTCAACTGCTTTGLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 459) (SEQ ID NO: 660)within exon 4705_CDS2_1 ACTGTTGCCATCATTGCTTC TCCAGACAGTACCACAGGAGA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 460) (SEQ ID NO: 661)within exon 5214_CDS14_1 GAGAAGCACGAGGAGTTCTGT GATAGTGTTCAGGGCGGTGT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 461) (SEQ ID NO: 662)within exon 55425_CDS7_1 GATGACTGAACTTCCTCCAGAA CCTTTCCCTATCAGCTGGAGTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 462) (SEQ ID NO: 663)within exon 9134_CDS11_1 TAGGAAGGAGCCACAGCATT TTGCCTTGCCATAACACATT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 463) (SEQ ID NO: 664)within exon 2260_RNA gaacaggcatgcaagtgaga gctgtagccctgaggacaag LungRNA only Real-time PCR (SEQ ID NO: 464) (SEQ ID NO: 665) 10160TCTCTAAGCCGCTTTCATCA TCCACGTTCCAAGGTACTGA Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 465) (SEQ ID NO: 666) within exon 1161ACTGAAATACACAGCAGTCAACAGT GGCTGCAGTTCAGAATTTGTT Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 466) (SEQ ID NO: 667) within exon 3248CTAGCCTTTGGTCCACATCA TCCTTCATGCACTTGGTGATA Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 467) (SEQ ID NO: 668) within exon 4256CGCTTCCTGAAGTAGCGATT CCTGTCCACGAGCTCAATAG Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 468) (SEQ ID NO: 669) within exon 5720GGCCCTGTAGGCTGTGTTAT GGGAGAGAGGGCTCTCAATA Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 469) (SEQ ID NO: 670) within exon 580CCTGACAGCTCATTGTCATGTA GACTGCATTGGAACTGGATG Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 470) (SEQ ID NO: 671) within exon 64122GATCCTTCTTCACCGCCTAC GGTTCCAGTGGTTCAGGTAGT Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 471) (SEQ ID NO: 672) within exon 80704GCCCAGATATTGGCTGTGTA GTGGGTTATGTGAAAGTCAACTG Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 472) (SEQ ID NO: 673) within exon 9169TTTGGAGCCTGTCTTGTTTG GAGCTGAAACAGCCAGTCAG Lung Both DNA_RNA_Real time PCR (SEQ ID NO: 473) (SEQ ID NO: 674) within exon 155GTGTCCGTGGGTCAACTTTT GTGACTGGATCCGGAAAGAA Lung Both DNA_RNA_Real-time PCR (SEQ ID NO: 474) (SEQ ID NO: 675) within exon137994_CDS0_1_RNA ATAGATGTGAAGCCCAAGCC TCAGTTGAGGTGCAAGATCC LungRNA only Real-time PCR (SEQ ID NO: 475) (SEQ ID NO: 676)54904_CDS0_2_RNA TTAACCGTCTCCTTGGTTCC TCCAGCCAAAACAATAGTGC Lung RNA onlyReal-time PCR (SEQ ID NO: 476) (SEQ ID NO: 677) 2017_RNATCTCCAAGCGGAAAGAAAGA CACAAAATCAGGGTCGGTCT Lung RNA only Real-time PCR(SEQ ID NO: 477) (SEQ ID NO: 678) 8500_RNA CCACATCTGTGCATGACCTCTTCCAAGCGCTCCTGTAACT Lung RNA only Real-time PCR (SEQ ID NO: 478)(SEQ ID NO: 679) 1978_RNA_DNA CTTCGTGAACACCAGCAGAT GAAGGAAGGGTTCGTTCTTGLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 479) (SEQ ID NO: 680)within exon 155_RNA_DNA CAGTGGTGCCTTACATGGTG TGGGAAGGTAGAGGTTGTGG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 480) (SEQ ID NO: 681)within exon nly aaaagaatccaggagcgaga aacacccacaacatcttcca Lung RNA onlyReal-time PCR (SEQ ID NO: 481) (SEQ ID NO: 682) y aacttattgggttggcgaagggacagacacacagcattcc Lung RNA only Real-time PCR (SEQ ID NO: 482)(SEQ ID NO: 683) y cagattttggctggtctgtg cactccaatgcaccacaaat LungRNA only Real-time PCR (SEQ ID NO: 483) (SEQ ID NO: 684) yaaactgctcaggcataaccc catcaggcgacagattgaag Lung RNA only Real-time PCR(SEQ ID NO: 484) (SEQ ID NO: 685) 598_RNA_only AGGACCAGGTGTTTGTCCTGAGGTGTGGTGCAGACACTTG Lung RNA only Real-time PCR  (SEQ ID NO: 485)(SEQ ID NO: 686) 1019_CDS1_1 TTGACTGTTCCACCACTTGTC TCTGATGCGCCAGTTTCTAALung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 486) (SEQ ID NO: 687)within exon 2735_CDS0_1 GTGCCGGAAGTCATACTCAC GGCATTGCTGAAGGCTTTAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 487) (SEQ ID NO: 688)within exon 1594_CDS2_1 CCAAAGATGTCTCTGCCTGA TCTGCCCTAGCCTGGTTTAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 488) (SEQ ID NO: 689)within exon 28954_CDS1_1 CAGAAGGATGTCCCATCTGA CAGGCCAGACCAGTCTACAG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 489) (SEQ ID NO: 690)within exon 598_CDS0_1 GGCCTCAGTCCTGTTCTCTT TGGACAATGGACTGGTTGAG LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 490) (SEQ ID NO: 691)within exon 853661_CDS0_1 CAGACTGCGACACCTGAGAC CTATGCAGAAAGGCAGGTGA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 491) (SEQ ID NO: 692)within exon 4216_CDS0_1 TTGCAAGAGTCCTGAATCTGA TGACATGCGTTTCATCTGTC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 492) (SEQ ID NO: 693)within exon 10846_CDS15_1 GCCGATACAGCTCGAGAATA CCAAGAGGTACTGGACTGGAATLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 493) (SEQ ID NO: 694)within exon 3482_CDS11_1 GGGAGACTCCTGTCGTCTGT GTGGAAAGGCTGGACAAGTT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 494) (SEQ ID NO: 695)within exon 3660_CDS0_1 GGAGATCTGCAGAGGGTAGAG TGGACAGCAACATTGAGAATC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 495) (SEQ ID NO: 696)within exon 3839_CDS2_1 CCGGCCATTATCAGAATGTT AGCAGAATGTAATACCACCGTTCLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 496) (SEQ ID NO: 697)within exon 84189_W_GENE_1 TCCTGGAAGTTGAGTGGATG CTGGCAGCAACATCTTCAATLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 497) (SEQ ID NO: 698)within exon 10326_CDS2_2 AGTTGTTACCCGTGGGAAGT ACTCTGCGCTGTGCTATGAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 498) (SEQ ID NO: 699)within exon 845_CDS0_1 GACACCGGCTCATGGTAGTA TATGATGGGAAGGACCGAGT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 499) (SEQ ID NO: 700)within exon 25960_CDS3_1 CATCACTCTGGCCTCATCAC TGTCTGTGCAGCAAGCATAA LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 500) (SEQ ID NO: 701)within exon 8749_CDS1_1 TGAACCAGTAGAATCTTCAGCAAG GGAACTTTGTAGGGCTGGTCTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 501) (SEQ ID NO: 702)within exon 65110_W_GENE_3 TTCTCCCGTAGTTGGGAAAG AGAGTCTCAGGG1TGGCACTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 502) (SEQ ID NO: 703)within exon 112399_CDS0_1 AGAGGCCATCTTTGGATCTC TGCCCTCACTGAAGACTGAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 503) (SEQ ID NO: 704)within exon 84684_W_GENE_1 ATTTCGAGGTTGGCCTTAGA ATTTCACTGTCCGCTCCTCTLung Both DNA_RNA_ Real-time PCR (SEQ ID NO: 504) (SEQ ID NO: 705)within exon 9054_exon3 ATGCCATGCTCCCTTACCTA CTGACGAGCACGTTCCAT LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 505) (SEQ ID NO: 706)within exon 8904_exon1 ATTTTGTGAAGCGGCGAAG GAACCCAGACCCCGAATTAC LungBoth DNA_RNA_ Real-time PCR (SEQ ID NO: 506) (SEQ ID NO: 707)within exon 3351_N_GENE_1 CTAGGGTCTVGGTGGCTTTG CCGGATCTCCTGTGTATGTG LungDNA only Real-time PCR (SEQ ID NO: 507) (SEQ ID NO: 708) 55023_CDS0_1TGCCCTATAGCTGTCAGTGTGT TAGTCGCTGCCTGAGAGTTG Lung DNA only Real-time PCR(SEQ ID NO: 508) (SEQ ID NO: 709) 134728_CDS0_1 AGCCTGTCTTGTTGCTGTGCTATTTGGCCTTCCCATTCT Lung DNA only Real-time PCR (SEQ ID NO: 509)(SEQ ID NO: 710) bp_BC001767 AAAAAACCCACCTACACGAT Lung DNA onlySequencing (SEQ ID NO: 711) bp_BC001767 ATCTCGAAGCAGAACCAAGT LungDNA only Sequencing (SEQ ID NO: 712) bp_BC001767 AAGGACACTTATAGGCTTTTLung DNA only Sequencing (SEQ ID NO: 510) bp_BC001767CGTGTAGGTGGGTTTTTTTTTA Lung DNA only Sequencing (SEQ ID NO: 511)WHSC1L1_seq_f_1 TGTGATCGCACTGACACGGC Lung RNA only Sequencing(SEQ ID NO: 512) WHSC1L1_seq_f_2 GGTACCACAAACTGTGATTC Lung RNA onlySequencing (SEQ ID NO: 513) WHSC1L1_seq-f_3 CCGAGAATATCATGTCCAGT LungRNA only Sequencing (SEQ ID NO: 514) WHSC1L1_seq_f_4TGCGGCAAGCAGGAAATCCT Lung RNA only Sequencing (SEQ ID NO: 515)WHSC1L1_seq_f_5 CCTCAACTGATGTAGAAAATGACTA Lung RNA only Sequencing(SEQ ID NO: 516) WHSC1L1_seq-f-6 AGATATCCACAAAGCAAGTA Lung RNA onlySequencing (SEQ ID NO: 517) WHSC1L1_seq_f_7 GTGGCCAGCAGAGATCTGCA LungRNA only Sequencing (SEQ ID NO: 518) WHSC1L1_seq_f_8ACAAAGAGACTATACCCTGA Lung RNA only Sequencing (SEQ ID NO: 519)WHSC1L1_seq_f_9 GAAGAGACGAAAGATCAAAA Lung RNA only Sequencing(SEQ ID NO: 520) WHSC1L1_seq_r_1 TTGCTTTFTTCACTTAAATA Lung RNA onlySequencing (SEQ ID NO: 521) WHSC1L1_seq_r_2 TAAGGCATAGGAGGTGGTAT LungRNA only Sequencing (SEQ ID NO: 522) WHSC1L1_seq_r_3ATCACAGAGAGCAAATAGTC9 Lung RNA only Sequencing (SEQ ID NO: 523)WHSC1L1_seq_r_4 ATGATCTCTGCATCAGGGTA Lung RNA only Sequencing(SEQ ID NO: 524) WHSC1L1_seq_r_5 CCTGGAAACGTTTTGCAGCT Lung RNA onlySequencing (SEQ ID NO: 525) WHSC1L1_seq_r_6 ACAATTCCAGCAGCCTTCTG LungRNA only Sequencing (SEQ ID NO: 526) WHSC1L1_seq_r_7GGAATGATTACTACAGATGAGA Lung RNA only Sequencing (SEQ ID NO: 527)WHSC1L1_seq_r_8 GCCCAGTTTTACATTCCATG Lung RNA only Sequencing(SEQ ID NO: 528) WHSC1L1_seq_r_9 TGTCTCTGTATGCTGAACTAG Lung RNA onlySequencing (SEQ ID NO: 529) WHSC1L1_seq_r_10 GGTGTAGAAATTATAAGCCT LungRNA only Sequencing (SEQ ID NO: 530) WHSC1L1_seq_r_11CGAATTTCAGTACTTGAGAG Lung RNA only Sequencing (SEQ ID NO: 531)WHSC1L1_seq_r_12 TTGCTGGCTTGTTTGGTTGC Lung RNA only Sequencing(SEQ ID NO: 532) WHSC1L1_seq_r_13 TCCTCTTTTAGTACTGGTTC Lung RNA onlySequencing (SEQ ID NO: 533) WHSC1L1_seq_r_14 TCTTTGGAATCACACAGTTTGT LungRNA only Sequencing (SEQ ID NO: 534) WHSC1L1_seq_r_15CTGGCCACCATCTTCAGCAA Lung RNA only Sequencing (SEQ ID NO: 535)LETM2_seq_f_1 GCCTTCTACAGTTATAATTCAG Lung RNA only Sequencing(SEQ ID NO: 536) LETM2_seq_f_2 GAGGAACAGAGCCAAGATGG Lung RNA onlySequencing (SEQ ID NO: 537) LETM2_seq_f_3 GACTGATATTCTTGTGGAAT LungRNA only Sequencing (SEQ ID NO: 538) LETM2_seq_r_1 AATTCAGTTCTGGCTATTGCLung RNA only Sequencing (SEQ ID NO: 539) LETM2_seq_r_2AGGAACAGAGCCAAGATGGG Lung RNA only Sequencing (SEQ ID NO: 540)LETM2_seq_r_3 TCTTGTGGAATTACCTACTT Lung RNA only Sequencing(SEQ ID NO: 541)

TABLE 7 List of high-confidence MCRs in lung AC and SCC primary tumorsand cell lines. MCR recurrence Minimal common regions (MCRs) Gain/ Amp/Max/Min No. of Loss Del Cytogenetic band Position (Mb) Size (Mb) valuetranscripts % T C T C Cancer-related genes miRNA Gain and amplification1p36.32-1p36.32 2.37-2.47 0.1 1.2 2 22 9 9 3 0 1p36.21-1p36.1215.29-20.69 5.4 1.32 86 20 8 9 1 1 SDHB, PAX7 1p34.3-1p34.3 37.82-38.130.32 2.92 11 18 7 8 1 1 1p34.2-1p34.1 41.65-44.9  3.26 2.35 50 19 6 10 32 1q21.3-1q22 150.95-151.92 0.98 1.96 29 49 17 24 3 13 1q31.2-1q32.1189.28-197.57 8.29 1.22 35 40 16 17 1 4 hsa-mir-181b-1, hsa-mir-2131q32.2-1q32.2 206.17-206.24 0.06 1.05 3 39 15 17 1 4 2p16.3-2p14 49.1-64.73 15.63 1.11 54 40 13 21 1 3 BCL11A, REL hsa-mir-217,hsa-mir-216 2q11.2-2q11.2 96.68-96.95 0.27 2.16 8 35 14 16 4 42q14.1-2q14.2 118.58-120.16 1.57 0.88 11 28 9 14 1 2 2q31.1-2q31.1170.19-170.32 0.13 1.14 4 28 11 13 1 1 3p12.1-3p11.1 87.07-88.45 1.381.32 8 17 6 8 1 1 3q11.2-3q12.3  95.26-103.06 7.8 0.82 43 33 15 12 2 1TFG 3q21.3-3q25.32 130.02-159.3  29.28 0.85 182 45 21 17 6 4 GMPS3q29-3q29 195.34-196.56 1.22 1.06 12 36 16 14 6 3 4q12-4q12 53.58-55  1.42 1.47 6 22 9 10 1 4 FIP1L1, CHIC2 4q13.3-4q13.3 75.33-76.03 0.7 0.926 17 7 7 2 1 4q21.1-4q21.1 77.09-78.06 0.97 0.85 13 14 5 7 1 15p15.33-5p15.33  0.53-0.92† 0.38 2.67 5 60 27 24 6 15 5p12-5p1242.76-43.48 0.73 2.69 9 52 23 20 3 13 5q31.3-5q31.3 140.55-140.66 0.121.99 11 19 6 10 2 4 6p25.2-6p24.3 2.96-8.04 5.09 1.59 33 39 16 16 2 26p22.3-6p22.3 17.87-20.56 2.69 1.59 9 36 15 15 2 3 DEK 6p22.1-6p22.127.95-28.47 0.52 0.91 24 33 13 14 1 1 6p12.3-6p12.2 49.91-52.16 2.251.21 6 35 13 16 2 2 hsa-mir-206, hsa-mir-133b 6p12.1-6p12.1 53.47-55.732.26 1.21 11 35 13 16 3 2 6q24.3-6q25.1 145.99-149.77 3.79 1.16 11 17 68 2 1 6q25.2-6q25.2 153.38-154.67 1.28 1.68 5 16 5 8 1 1 7p11.2-7p11.254.41-55.94 1.53 3.62 15 36 13 18 2 7 EGFR 7q21.3-7q21.3  93.2-95.37†2.17 1.96 13 40 11 22 2 7 7q21.3-7q22.1  97.13-98.66† 1.54 1.96 17 40 1023 2 7 7q31.2-7q31.2 115.22-116.9  1.68 2.15 10 34 10 18 2 7 MET8p12-8p11.22*  38.24-38.45*† 0.21 1.98 2 35 17 12 6 4 WHSC1L1, FGFR18q11.21-8q22.1  48.86-97.23† 48.37 1.42 192 52 19 24 5 11 NCOA2, NBSI,hsa-mir-124a-2 CBFA2T1 8q24.13-8q24.13  123.37-126.14† 276 0.9 23 54 2124 4 9 8q24.21-8q24.21  128.5-129.02† 0.52 4.36 3 59 22 27 4 14 MYC8q24.3-8q24.3 145.67-146.25 0.58 0.81 21 52 20 23 4 8 RECQL410q24.1-10q24.1 97.62-98.11 0.49 1.15 8 16 3 10 1 1 10q26.3-10q26.3135.24-135.24 0.01 1.28 1 11 1 8 1 3 11p14.2-11p14.1 26.54-28   1.460.95 13 20 7 10 1 1 11p13-11p13 31.76-34.08 2.32 2.84 21 23 9 10 1 1WT1, LMO2 11q13.3-11q13.3  69.3-69.99 0.69 3.46 8 37 11 21 3 611q22.1-11q22.2  99.73-102.15 2.41 0.94 16 27 9 13 1 3 BIRC312p13.33-12p13.33 0.17-0.81 0.64 1.09 7 36 12 18 1 6 12p13.2-12p12.312.37-16.6  4.22 1.16 39 29 11 13 2 5 12p12.1-12p11.23  24.61-26.95†2.34 1.98 13 37 15 16 5 7 KRAS2 12q12-12q13.11 43.74-46.42 2.68 2.68 1431 11 15 2 6 12q13.2-12q13.2 54.72-54.84 0.12 1.4 8 29 10 14 2 2 ERBB312q15-12q15 67.42-67.95 0.53 1.82 5 34 14 14 5 4 13q32.1-13q32.194.07-95.21 1.14 1.03 6 6 3 2 1 1 13q34-13q34  111.77-112.97† 1.2 1.2116 14 4 8 2 5 13q34-13q34 113.25-114.11 0.86 1.12 10 14 4 8 1 514q13.2-14q13.3  34.64-36.22† 1.58 2.23 20 29 9 15 2 4 14q22.2-14q22.353.49-55.15 1.66 1.41 22 30 8 17 1 6 14q32.13-14q32.13 93.66-94.03 0.371.1 9 29 6 18 1 4 15q25.2-15q25.3 82.35-83.48 1.13 0.95 22 31 10 16 2 516p12.1-16p12.1 23.68-27.37 3.69 0.89 21 18 6 9 1 2 16q12.1-16q12.251.03-52.87 1.84 1.3 9 20 7 10 1 4 16q22.2-16q22.2 69.62-69.87 0.26 1.082 18 6 9 1 4 16q24.2-16q24.3 86.43-87.23 0.81 1.7 9 18 5 10 2 317p11.2-17p11.2 17.09-18.86 1.77 0.84 40 20 7 10 1 1 18p11.32-18p11.320.2-0.9 0.7 1.7 9 25 13 9 4 1 18q11.2-18q11.2 18.25-20.9† 2.65 0.96 1520 8 9 2 0 18q12.1-18q12.1 26.83-27.31 0.48 1.55 6 19 9 7 2 118q12.3-18q21.1 37.92-46.03 8.12 1.41 33 23 11 8 1 1 19p13.12-19p13.12 14.8-16.01 1.22 1.53 39 11 5 4 1 1 BRD4 19q12-19q12 35.01-35.73 0.731.91 4 31 13 13 2 5 19q13.33-19q13.33  55.52-55.68† 0.16 1.01 8 17 5 9 11 20p12.3-20p12.2  7.91-10.57 2.66 0.85 12 31 13 14 1 320q11.21-20q11.21  29.66-29.88† 0.22 3.85 5 42 13 23 1 820q13.32-20q13.33  57.04-60.21† 3.17 1.61 19 41 12 23 1 11 SS18L121q11.2-21q21.1 14.52-18.56 4.04 1.14 13 17 7 7 2 0 hsa-mir-99a,hsa-let-7c, hsa-mir-125b-2 22q11.21-22q11.21 19.09-20.3  1.22 1.02 28 2511 10 1 3 Loss and deletion 1p22.1-1p22.1 92.45-94.07 1.62 −0.89 15 18 97 1 1 2q34-2q35 209.15-217.17 8.02 −0.83 25 11 6 3 1 1 AT1C2q36.1-2q37.1 223.74-230.87 7.12 −1.03 28 8 5 2 1 1 3p26.3-3p26.20.43-3.09 2.66 −0.87 4 23 11 9 1 3 3p11.2-3p11.1 87.39-88.45 1.07 −0.926 24 14 7 3 1 4p15.32-4p15.32 15.84-17.48 1.64 −0.88 8 14 8 5 2 06p21.32-6p21.32 32.47-33.01 0.54 −1.05 17 11 3 6 2 1 6q21-6q22.33 111.4-128.94† 17.55 −1 72 23 8 11 1 4 7p22.3-7p22.3 1.36-2.05 0.69−1.21 7 10 5 3 1 1 7q34-7q34 142.28-142.44 0.16 −1.39 4 13 7 4 2 18p21.1-8p12  28.74-33.48† 4.74 −1.28 22 36 15 16 1 8 WRN 9p21.3-9p21.321.47-22†   0.53 −4.81 4 43 16 21 1 18 CDKN2A hsa-mir-31 11q11-11q11^(†) 55.1-55.18 0.08 −4.06 2 23 13 6 7 1 11q24.3-11q24.3 128.13-129.24 1.11−0.84 9 16 5 8 1 4 12q12-12q13.11  42.41-44.87† 2.46 −1.94 13 7 2 4 1 213q12.11-13q12.11 19.14-19.56 0.41 −3.51 5 34 14 14 1 7 13q14.13-13q21.145.82-57.2  11.38 −1.35 60 35 16 13 1 8 RB1 hsa-mir-16-1, hsa-mir-15a13q32.2-13q32.2 97.47-97.9  0.42 −3.25 3 29 13 11 1 8 18q21.33-18q22.2 58.4-65.82† 7.43 −1.26 26 29 10 14 1 5 21p11.2-21p11.1  9.93-10.08†0.15 −1.33 3 17 9 6 1 1 The numbers of primary tumors (T) or cell lines(C) with gain or loss and amplification or deletion are listed,respectively See text for other definitions MCR recurrence is denoted asthe percentage of the total dataset In bold are the MCRs verified byRT-PCR and FISH (FIGS. 2 and 7-10, data not shown) Black diamondsidentify high-priority MCRs that are in common with the PDAC dataset(1). The short arm of chromosome 3 was consistently lost across severalprimary tumors and cell lines (FIGS. 1 and 6) Three smaller regionswithin the short arm of chromosome 3 were identified by the automatedlocus definition program, based on the presence and recurrence ofdeletions in a subset of samples (FIG. 6, bright green bars). The numberof transcripts is based on Build 35, National Center for BiotechnologyInformation Only the known genes within the boundaries have beenincluded The list of cancer-related genes was derived from Futreal etal. (3) *MCR in 8p, which was subject to further fine mapping (see text)†The MCR at 11q11 has recently been shown by Sebat et al (2) to be acopy-number polymorphism (ORF5I1 on chromosome 11q11) 1. Aguirre, A J.,Brennan, C., Bailey, G, Sinha, R, Feng, B, Leo, C, Zhang, Y., Zhang, J.,Gans, J. D, Bardeesy, N, et al. (2004) Proc. Natl. Acad. Sci USA 101,9067-9072 2. Sebat, J., Lakshmi, B., Troge, J, Alexander, J, Young, J.,Lundin, P., Maner, S, Massa, H., Walker, M., Chi, M., et al. (2004)Science 305, 525-528. 3. Futreal, P. A., Coin, L., Marshall, M., Down,T, Hubbard, T, Wooster, R., Rahman, N & Stratton, M. R. (2004) Nat. RevCancer 4, 177-183

TABLE 8 Affymetrix Probe Cytogenetic band Position NCBI Gene Name Genefull name 209863_s_at 3q28 190, 831, 918 TP73L tumor protein p73-like211194_s_at 3q28 190, 831, 918 TP73L tumor protein p73-like 211195_s_at3q28 190, 831, 918 TP73L tumor protein p73-like 218182_s_at 3q28 191,506, 196 CLDN1 claudin 1 222549_at 3q28 191, 506, 196 CLDN1 claudin 11552291_at 3q29 197, 927, 625 PIGX phosphatidylinositol glycan, class X202514_at 3q29 198, 258, 815 DLG1 discs, large homolog 1 (Drosophila)

TABLE 9 Markers of the Invention displaying increased expression in LungCancer Primary Tumors. Gene Symbol Chromosome Entrez ID (A) NPC1 18 4864BRD9 5 65980 CHCHD2 7 51142 CAS1 7 64921 BRI3 7 25798 TMEM16F 12 196527AMIGO2 12 347902 MBC2 12 23344 NIT2 3 56954 RG9MTD1 3 54931 TRRAP 7 8295RAB2 8 5862 RNF139 8 11236 NDUFB9 8 4715 SPC18 15 23478 RIOK3 18 8780CPOX 3 1371 ASNS 7 440 PSMA6 14 5687 GCH1 14 2643 LCMT1 16 51451 ATP5E20 514 PSMA7 20 5688 HTPAP 8 84513 PON2 7 5445 (B) Whsc1l1 8 54904 Brd419 23476 ss18l1 20 26039 CAMK1G 1 57172 CNNM4 2 26504 CNNM3 2 26505 PPIG2 9360 tpx2 20 22974 BRD9 5 65980 SEMA4C 2 54910 CCNI 4 10983

1. A method of treating a subject afflicted with cancer comprisingadministering to the subject a modulator that specifically modulates theamount and/or activity of Wolf-Hirschhorn syndrome candidate 1-like 1(WHSC1L1), wherein the modulator is a nucleic acid molecule that iscomplementary to WHSC1L1, thereby treating a subject afflicted withcancer.
 2. A method of treating a subject afflicted with cancercomprising administering to the subject a compound which specificallyinhibits the amount and/or activity of WHSC1L1 which is amplified incancer, wherein the compound is a nucleic acid molecule that iscomplementary to WHSC1L1, thereby treating a subject afflicted withcancer.
 3. The method of claim 2, wherein said compound is administeredin a pharmaceutically acceptable formulation.
 4. The method of claim 2,wherein said compound is an RNA interfering agent which inhibitsexpression of WHSC1L1.
 5. The method of claim 4, wherein said RNAinterfering agent is an siRNA molecule or an shRNA molecule.
 6. Themethod of claim 2, wherein said compound is an antisense oligonucleotidecomplementary to WHSC1L1.
 7. The method of claim 2, wherein saidcompound is an aptamer which inhibits expression or activity of WHSC1L1.